Methods and compositions for achieving hemostasis and stable blood clot formation

ABSTRACT

Provided is tunable biopolymer hydrogel produced from two processed natural polysaccharides for use as a hemostat. If desired, the hydrogel formation can be tuned so that the hydrogel forms within seconds when applied to a tissue lesion. The resulting hydrogel can adhere to tissue and, without swelling, produce hemostasis within seconds after application to tissue of interest. The hydrogel also captures, aggregates and concentrates platelets and red blood cells at the site of the tissue lesion thereby initiating a clotting cascade at the site of the lesion. The hemostat can be used to prevent blood loss during surgical procedures, for example, during brain, spine or other surgical procedures where hemostasis is desirable, and is particularly useful during surgical procedures where swelling of the hemostat (e.g., in the brain or spine) would be detrimental to the subject.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/194,917, filed Nov. 19, 2018, the entire disclosure of which isincorporated herein by reference for all purposes.

FIELD

The invention relates generally to methods and compositions forachieving hemostasis and blood coagulation, and more specificallyrelates to methods and compositions for achieving hemostasis and bloodcoagulation during surgical procedures.

BACKGROUND

Bleeding and blood loss are problems that must be addressed during andat the conclusion of surgery and other medical procedures. Significantblood loss can be fatal or cause significant morbidity, and surgeons andmedical staff routinely compensate for blood loss with transfusions andthe use of recirculating devices. A number of different techniques andproducts are currently used by surgeons to stop bleeding and effecthemostasis in surgery and medical procedures. However no single approachis effective or suitable under all circumstances, and the currentlyavailable approaches have limitations in effectiveness, safety,applicability and ease of use.

The most commonly used approaches involve closing a tissue or bloodvessel mechanically with a suture or staple. This ligature technique isoften effective, though it may allow for minor bleeding to occur aroundsuture or staple holes. Sometimes biological or synthetic adhesives areapplied to either complement or replace sutures or staples to stop thisblood flow. However, tissue sealants are not hemostats per se and allowthe pooling of blood underneath the seal in cases of moderate bleedingthat may result in the formation of a hematoma. Hemostatic productswhich require time-consuming application or which produce slow effectiveaction can be costly both in terms of financial costs due to greateroperating room time and in terms of post procedure patient morbidity.Some techniques and products have limited effectiveness in certainapplications, which may require a surgeon to reapply the product orswitch to another product, which can be both time consuming and costly.

Under certain circumstances standard ligature techniques areinappropriate or not feasible. In such cases, an absorbable hemostaticproduct can be applied to the bleeding surface with a goal of achievinghemostasis that will later lead to a durable clot formation. Passivehemostats can control bleeding through absorption and may be powders,gauze, sponges or cross-linked gelatin. Sometimes these are augmentedwith an active hemostatic agent such as thrombin to try to achieve asuccessful hemostasis. However, the efficacy of these products is oftenvariable and their use has been associated with adverse effects whichcan provide significant challenges during a surgical procedure. Suchchallenges become especially pronounced in certain surgical proceduressuch as brain and spinal surgeries. For example, certain sponges derivedfrom porcine material can absorb up to 45-times their weight in bloodand fluids resulting in significant swelling of the product that canlead to a patient experiencing adverse reactions such as pain, paresisor paralysis that can require revision surgery to address. Certainparticulate hemostats have been reported to have caused cerebral edema.Gelatin-based devices typically swell during use and have been reportedto cause mass effects leading to the development and progression ofhemiparesis that required revision surgery to address the situation.Other adverse events and complications have been reported for all thesetypes of hemostats. Most of these devices rely on absorption of bloodand body fluids to produce hemostasis, which results in swelling and canlead to the reported mass effects.

The slow action of available absorbable hemostats, which are often usedmultiple times per case, can add to the length of surgical procedures,adding to expense and post-operative morbidity. Certain commerciallyavailable hemostats require as much as 10 minutes to produce hemostasisfor each incidence of bleeding during a surgical procedure.

Accordingly, there remains a need for a fast acting, ready to use,biocompatible, biodegradable hemostat that minimizes or preventsbleeding and blood loss, does not swell during use and promotes stableclot formation during a wide variety of surgical procedures includingcranial and spinal surgeries and in minimally invasive surgicalprocedures, and significantly reduces the incidence of postoperativeadverse events.

SUMMARY OF THE INVENTION

The invention is based upon the discovery that it is possible to createa fast acting biocompatible hemostat that, promotes coagulation,produces hemostasis, does not swell during use, is adherent and does notmigrate away from a site of application, is transparent, and isbiodegradable.

The invention provides a “tunable” biopolymer system that addressesheretofore unmet needs in the field of hemostasis. In this system, twoprocessed natural polysaccharides in the form of separate solutions aresimultaneously mixed and applied onto a bleeding site, where they form ahydrogel device in situ. The hydrogel can form and produces hemostasiswithin seconds after application. The system may also be designed or“tuned” to produce hydrogels and facilitate hemostasis more slowlydepending on the specific method of use. Rapid hemostasis can alsocontribute to the reduction of major complications in some surgicalprocedures by facilitating a shorter procedure time. It is believed thatthe rapid hemostasis acts to quickly stop bleeding and the hydrogelcomprises specific intrinsic properties resulting in the simultaneouscapture, aggregation and concentration of platelets and red blood cellsto initiate the cascade of coagulation, which is accomplished withoutthe use of exogenously added clotting agents, drugs or other chemicals.The resulting hemostatic action assures that the device does not actsimply as a tamponade or sealant that might allow the pooling of bloodand formation of a hematoma under a sealant.

The hydrogel itself forms without the use of a third chemical orsynthetic cross-linking agent and thus avoids the potential forirritation and cytotoxicity or the interference with predictable rapidbiodegradation associated with cross-linking agents. Furthermore, due tothe internal chemistry, the hydrogel itself gently and predictablycontracts over time and does not swell, even when immersed in blood orbody fluids. This feature permits the hemostats described herein to beused in brain, spinal or other surgical procedures in which swelling ofa hemostatic agent can lead to mass effects and subsequentcomplications, which themselves require medical or surgicalintervention.

Furthermore, the hydrogel provided herein is essentially transparent, sothat a surgeon can visually verify its action and effect in real time.This feature allows surgeons to proceed with the surgical procedure andclose the site while verifying hemostasis, in contrast to hemostatswhich are opaque.

Furthermore, the hydrogel provided herein is cohesively strong and thuscan quickly establish its initial tamponade effect. Furthermore, thehemostat is also adherent and does not migrate away from the applicationsite. The hydrogel forms in intimate continuous contact with thebleeding site so that all sources of bleeding are exposed and treated tothe hemostatic action of the device.

The hemostatic hydrogel is produced by combining a defined acrylatedchitosan composition as described herein to a defined oxidized dextrancomposition also described herein to produce a hemostatic hydrogelhaving the structural and/or functional properties discussed herein. Thecharacteristics of the acrylated chitosan and oxidized dextran togetherwith their respective syntheses required to produce a hemostatichydrogel having the desired properties are discussed below.

In one aspect, the invention provides an acrylated chitosan compositionfor use in creating the hemostat described herein, wherein the acrylatedchitosan composition comprises acrylated chitosan comprising:

-   -   (i) from about 0.01 to about 0.3 mole fraction of a first        monomer of formula (I)

-   -   (ii) from about 0.3 to about 0.75 mole fraction of a second        monomer of formula (II)

and

-   -   (iii) from about 0.2 to about 0.7 mole fraction of a third        monomer of formula (III)

In certain embodiments, the acrylated chitosan composition comprises (i)from about 0.01 to about 0.26 mole fraction of the first monomer offormula (I), (ii) from about 0.35 to about 0.65 mole fraction of thesecond monomer of formula (II), (iii) from about 0.3 to about 0.6 molefraction of the third monomer of formula (III), or a combination of (i)and (ii), (i) and (iii), (ii) and (iii), and (i), (ii) and (iii). Incertain embodiments, the acrylated chitosan comprises less than about0.1 mole fraction of a fourth monomer of formula (IV)

In each of the foregoing, the acrylated chitosan can have (i) aweight-average molecular weight (Mw) of from about 25 kDa to about 400kDa or from about 115 kDa to about 200 kDa, (ii) a number-averagemolecular weight (Mn) of from about 14 kDa to about 200 kDa or fromabout 55 kDa to about 140 kDa, (iii) a polydispersity index (PDI) offrom about 1.5 (Mw/Mn) to about 3.5 (Mw/Mn), (iv) a PDI of from about1.5 (Mz/Mw) to about 7.0 (Mz/Mw), or from about 1.6 (Mz/Mw) to about 3.0(Mz/Mw) or a combination of two, three or four of each of the foregoingfeatures.

In certain embodiments, the acrylated chitosan composition comprisesfrom about 1% (w/w) to about 25% (w/w), or from about 1% (w/w) to about10% (w/w). In other embodiments, the acrylated chitosan composition hasa viscosity of from about 10 cP to about 50,000 cP, or from about 100 cPto about 25,000 cP.

In another aspect, the invention provides a method of preparing anacrylated chitosan composition (for example, the acrylated chitosancompositions described above) comprising the steps of (a) contacting araw chitosan material with an acetic acid solution (e.g., a 1% (v/v)acetic acid solution) to form a chitosan intermediate; (b) contactingthe intermediate chitosan material with acrylic acid to form anacrylated chitosan intermediate; and (c) purifying the acrylatedchitosan intermediate to produce an acrylated chitosan composition ofthe present invention.

In certain embodiments, during step (a), the step of contacting the rawchitosan material with the acetic acid solution is conducted at (i) apressure of about 2 atmospheres, (ii) a temperature from about 80° C. toabout 120° C., or a combination thereof. In certain embodiments, duringstep (a), (i) the PDI (Mw/Mn) of the chitosan intermediate is from about15% to about 40% less than the PDI (Mw/Mn) of the raw chitosan material,(ii) the raw chitosan material has a PDI of from about 1.5 (Mw/Mn) toabout 5.0 (Mw/Mn), (iii) the raw chitosan material has a degree ofdeacetylation of from about 65% to about 99% or from about 70% to about98%, or a combination of (i) and (ii), (i) and (iii), (ii) and (iii),and (i), (ii) and (iii).

In certain embodiments, the during step (b), (i) the first chitosanintermediate is contacted with the acrylic acid at a temperature fromabout 50° C. to about 120° C., (ii) the acrylated chitosan intermediatehas a degree of substitution (DS) value of from about 25% to about 65%,or a combination of (i) and (ii). In certain embodiments, the methodfurther comprises the step of precipitating the acrylated chitosancomposition to provide a solid and then optionally or in additiondissolving the solid into an aqueous medium to form a solutioncomprising an amount of the acrylated chitosan composition. In certainembodiments, the acrylated chitosan composition comprises from about 1%to about 25%, or from about 1% to about 10% by weight of the totalweight of the solution.

In another embodiment, the invention provides a method of preparing anacrylated chitosan composition comprising the steps of: (a) contacting araw chitosan material with an acetic acid solution to form a chitosanintermediate, wherein the raw chitosan material has a PDI of from about1.5 (Mw/Mn) to about 5.0 (Mw/Mn); (b) contacting the intermediatechitosan with acrylic acid to form an acrylated chitosan intermediatehaving a degree of substitution (DS) value of from about 25% to about65%; and (c) purifying the acrylated chitosan intermediate to produce anacrylated chitosan composition of the present invention. The method mayfurther comprise the step of precipitating the acrylated chitosancomposition produced in step (c) to provide a solid and then optionallyor in addition dissolving the solid into an aqueous medium to form asolution comprising from about 1% to about 10% acrylated chitosancomposition (w/w) of the total weight of the solution.

In another aspect, the invention provides an oxidized dextrancomposition for use in creating the hemostat described herein, whereinthe oxidized dextran composition comprises oxidized dextran comprising:

-   -   (a) less than about 0.8 mole fraction of a first monomer of        formula (V)

and

-   -   (b) from about 0.1 to about 1.0 mole fraction of a second        monomer, wherein the second monomer is selected from a monomer        of formula (VI), a monomer of formula (VII) and a combination of        formula (VI) and formula (VII)

In certain embodiments, the oxidized dextran composition comprises (i)from about 0.4 to about 0.7 mole fraction of the first monomer offormula (V), (ii) from about 0.15 to about 0.35 mole fraction of thesecond monomer, (iii) comprises less than about 0.65 mole fraction of athird monomer of formula (VIII)

or a combination of (i) and (ii), (i) and (iii), (ii) and (ii), or (i),(ii) and (iii).

In certain embodiments, the oxidized dextran has (i) a Mw of from about10 kDa to about 300 kDa or from about 15 kDa to about 90 kDa, (ii) a Mnof from about 4 kDa to about 166 kDa or from about 4 kDa to about 45kDa, (iii) a PDI of from about 1.8 (Mw/Mn) to about 6.0 (Mw/Mn) or fromabout 2.0 (Mw/Mn) to about 4.0 (Mw/Mn), (iv) a PDI of from about 1.5(Mz/Mw) to about 6.0 (Mz/Mw) or from about 1.5 (Mz/Mw) to about 5.0(Mz/Mw), or a combination of any of the foregoing features.

In certain embodiments, the oxidized dextran comprises a total amount ofaldehyde groups of from about 0.5 to about 2.0 or from about 0.6 toabout 0.9 mol aldehydes/mol oxidized dextran. In other embodiments, theoxidized dextran contains a ratio of primary aldehyde groups tosecondary aldehyde groups from about 1.8 to about 6.0 or from about 1.8to about 3.5. In certain other embodiments, the oxidized dextrancomposition has a degree of oxidation from about 25% to about 100% orfrom about 30% to about 50%. In certain other embodiments, the oxidizeddextran composition comprises from about 1% (w/w) to about 25% (w/w), orfrom about 1% (w/w) to about 10% (w/w) of the oxidized dextran. Incertain other embodiments, the oxidized dextran composition has aviscosity of from about 1 cP to about 2,000 cP, or from about 1 cP toabout 100 cP, from about 1 cP to about 10 cP.

In another aspect, the invention provides a method of preparing anoxidized dextran composition (for example, any of the oxidized dextrancompositions described herein), the method comprising the steps of: (a)contacting a raw dextran material with an oxidizing agent to form anoxidized dextran intermediate; and (b) purifying the oxidized dextranintermediate to produce an oxidized dextran composition of the presentinvention.

In certain embodiments, the raw dextran material has (i) a Mw of fromabout 50 kDa to about 2,000 kDa, (ii) a PDI of from about 1.4 (Mw/Mn) toabout 5.0 (Mw/Mn), or a combination of (i) and (ii). In certainembodiments, the oxidizing agent is a periodate salt. In certainembodiments, the method comprises the additional step of precipitatingthe oxidized dextran composition to provide a solid and then optionallyor in addition comprises the step of dissolving the solid into anaqueous medium to form a solution comprising an amount of the oxidizeddextran composition. In certain embodiments, the amount of the oxidizeddextran composition is from about 1% (w/w) to about 25% (w/w), or fromabout 1% (w/w) to about 10% (w/w) by weight of the total weight of thesolution.

In another aspect, the invention provides a hemostatic hydrogelcomposition containing oxidized dextran and acrylated chitosan, whereinthe hydrogel composition has two or more of the following features:

-   -   (i) the hydrogel composition comprises a total amount of free        aldehyde groups of from about 0.1 to about 0.7 moles        aldehyde/mole oxidized dextran,    -   (ii) the hydrogel composition is formed from oxidized dextran        and acrylated chitosan, wherein the ratio of primary aldehydes        in the oxidized dextran to the amines in the acrylated chitosan        is in the range from about 1.0 to about 2.0, and/or the ratio of        total aldehydes in in the oxidized dextran to amines in the        acrylated chitosan is from about 1.5 to about 3.0,    -   (iii) the ratio of Mw of the acrylated chitosan to the oxidized        dextran is from about 2 to about 10 the ratio of Mn of the        acrylated chitosan to the oxidized dextran is from about 4 to        about 15, the ratio of Mz of the acrylated chitosan to the        oxidized dextran is from about 2 to about 10, the ratio of PDI        (Mw/Mn) of acrylated chitosan to oxidized dextran is from about        0.5 to about 0.8, and/or the ratio of PDI (Mz/Mw) of acrylated        chitosan to oxidized dextran is from about 0.5 to about 1.0,    -   (iv) upon formation of the hydrogel composition, the hydrogel        composition comprises a bound water content of from about 65%        w/w to about 95% w/w,    -   (v) the hydrogel composition comprises a three-dimensional        porous structure comprising layers of substantially        non-interconnected pores having (a) a pore size distribution        from about 10 μm to about 850 μm in diameter, (b) a platelet        adhesive surface, or a combination of (a) and (b),    -   (vi) the hydrogel composition comprises walls disposed between        the substantially non-interconnected pores, the walls having a        wall thickness of from 0.046 μm to 50 μm,    -   (vii) the hydrogel composition comprises pores certain of which        define a platelet adhesive surface so that, when in contact with        blood, the hydrogel composition permits platelets and/or red        blood cells within the blood to adhere to the platelet adhesive        surface and promote blood clot formation at or within the        hydrogel composition,    -   (viii) the hydrogel composition comprises pores certain of which        define a platelet adhesive surface so that, when in contact with        blood, the hydrogel composition permits platelets and/or red        blood cells within the blood to adhere to the platelet adhesive        surface and not permit platelets and/or red blood cells from the        blood to enter pores present in a first surface of the hydrogel        composition, pass through the hydrogel composition, and then        exit the hydrogel composition via pores present in a second        surface of the hydrogel composition that opposes the first        surface,    -   (ix) at about 10 seconds after the formation of the hydrogel        composition, the hydrogel composition has a burst strength of        greater than 20 mmHg as determined using an ASTM F 2392-04        protocol,    -   (x) at about 2 minutes after the formation of the hydrogel        composition, the hydrogel composition has a burst strength of        greater than about 35 mmHg as determined using an ASTM F 2392-04        protocol,    -   (xi) at about 5 minutes after the formation of the hydrogel        composition, the hydrogel composition has a burst strength of        greater than about 70 mmHg as determined using an ASTM F 2392-04        protocol,    -   (xii) the hydrogel composition has an elastic modulus of from        about 500 Pa to about 5000 Pa at from about 10 seconds to about        80 seconds after the formation of the hydrogel composition,    -   (xiii) the hydrogel composition has a compression modulus of        from about 3 kPa to about 250 kPa,    -   (xiv) the hydrogel composition has an average adhesion strength        of from about 1.0 N to about 50 N, as determined using an ASTM F        2258-05 protocol,    -   (xv) the volume of the hydrogel composition, when formed, does        not increase upon exposure to a physiological fluid or body        fluid,    -   (xvi) the volume of the hydrogel composition shrinks by less        than about 5% about 10 minutes after formation when exposed to a        physiological fluid or body fluid,    -   (xvii) the hydrogel composition is substantially transparent        when the hydrogel composition has a thickness of 2 mm to 10 mm,        and    -   (xviii) the hydrogel composition optionally further comprises a        therapeutic agent.

In certain embodiments, the hemostatic hydrogel composition comprises:

-   -   (a) from about 0.0 to about 0.3 mole fraction of a first monomer        of formula (I)

-   -   (b) from about 0.02 to about 0.7 mole fraction of a second        monomer of formula (III),

and

-   -   (c) from about 0.0 to about 0.8 mole fraction of a second        monomer of formula (V),

In certain embodiments, the hemostatic hydrogel composition of comprises(i) from about 0.0 to about 0.26 mole fraction of the first monomer offormula (I), (ii) from about 0.03 to about 0.6 mole fraction of thethird monomer of formula (III), (iii) from about 0.04 to about 0.7 molefraction of the first monomer of formula (V), or a combination or (i)and (ii), (i) and (iii), (ii) and (iii) and (i), (ii) and (iii).

In certain embodiments, the hemostatic hydrogel composition comprises(i) no greater than about 0.2 mole fraction of a fourth monomer offormula (IV)

(ii) no greater than about 0.65 mole fraction of a fifth monomer offormula (VIII)

or a combination or (i) and (ii).

In certain embodiments, the hemostatic hydrogel comprises a plurality ofcrosslinked moieties of formula (IX)

In another aspect, the invention provides a method of preparing ahemostatic hydrogel composition (such as the hemostatic hydrogelcompositions described herein), the method comprising contacting anacrylated chitosan composition as described herein with an oxidizeddextran composition as described herein so as to form the hemostatichydrogel composition.

In certain embodiments, the ratio of the viscosity of the acrylatedchitosan composition to the oxidized dextran composition ranges fromabout 100:1 to about 10,000:1. In certain embodiments, the hemostaticagent further comprises a therapeutic agent or a cell, for example, astem cell.

In certain embodiments, the hemostatic hydrogel is produced bycontacting the acrylated chitosan composition described herein with theoxidized dextran composition described herein in a static mixer andoptionally or in addition then aerosolizing the mixture prior toapplication to tissue. In certain embodiments, the gelation time of thehemostatic hydrogel composition ranges within from about 10 seconds toabout 240 seconds after contacting the acrylated chitosan compositionwith the oxidized dextran composition.

In another aspect, the invention provides a method of promotinghemostasis (e.g., reducing or stopping blood loss) at a location in asubject in need thereof. The method comprises forming at the location inthe subject the hemostatic hydrogel composition described herein therebyto promote hemostasis (e.g., reduce or stop blood loss) at the location.It is understood that the hemostatic hydrogel composition adheres to atissue surface at the location. Furthermore, it is understood that thehydrogel permits the platelets and/or red blood cells to adhere to orotherwise become disposed on the surfaces of pores in the hemostatichydrogel and promote a blood coagulation cascade at the location therebyto produce a blood clot. It understood that blood loss at the locationcan be caused by, for example, trauma, abrasion, or surgicalintervention at the location.

The hemostatic composition, when applied to the location, can fill acavity at the location without inducing compression of tissuesurrounding the cavity when the hemostatic composition is exposed tophysiological fluid or a body fluid. It is understood that hemostasiscan be achieved from about 5 seconds to about 120 seconds, from about 10seconds to about 120 seconds, from about 15 seconds to about 120seconds, from about 5 seconds to about 60 seconds, from about 10 secondsto about 60 seconds, from about 15 seconds to about 60 seconds, fromabout 5 seconds to about 30 seconds, from about 10 seconds to about 30seconds, from about 15 seconds to about 30 seconds, from about 5 secondsto about 15 seconds, or from about 10 seconds to about 15 seconds, afterthe hemostatic hydrogel composition is applied to the location.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a scanning election microscope (SEM) image of the internalthree-dimensional structure of an exemplary acrylated chitosan/oxidizeddextran hydrogel.

FIG. 2 is a SEM image of the surface of an exemplary acrylatedchitosan/oxidized dextran hydrogel.

FIG. 3 is a graph showing the elastic modulus of an exemplary acrylatedchitosan/oxidized dextran hydrogel as a function of time.

FIG. 4 is a graph showing the weight of an exemplary acrylatedchitosan/oxidized dextran hydrogel following formation of a hydrogel asa function of time.

FIG. 5 is an SEM image of a blood clot formed within a pore of anexemplary acrylated chitosan/oxidized dextran hydrogel followingformation of a hydrogel at an abrasion wound on a porcine liver.

FIG. 6 is an SEM image of a blood clot formed within a pore of anexemplary acrylated chitosan/oxidized dextran hydrogel followingformation of a hydrogel at a punch biopsy wound on a porcine liver.

FIG. 7 is an SEM image of a blood clot formed within a pore of anacrylated chitosan/oxidized dextran hydrogel following formation of ahydrogel at a laceration on a porcine liver.

FIG. 8 is an SEM image of a blood clot formed on an acrylatedchitosan/oxidized dextran hydrogel following formation of a hydrogel ata laceration on a porcine spleen.

DETAILED DESCRIPTION OF THE INVENTION I Definitions

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below.

The terms “a” and “an” as used herein mean “one or more” and include theplural unless the context is inappropriate.

As used herein, the term “tissue” refers to material that forms thesolid or semi-solid structures that make up any of the organs orcomponents of a living organism and includes, for example, membrane,skin, muscle, bone, cartilage, nerve and nerve sheathe, meninge,connective tissue, blood vessel, the sclera or iris of the eye, thesolid materials constituting internal organs such as liver, stomach,pancreas, intestine, kidney, thymus, uterus, testes, bladder, lung,heart and any other internal structures that are solid or semi-solid intexture. Thus, liquids such as blood are not considered to be a tissue.

“Adhere” or “adherence” refers to the creation of a physical bondbetween material and tissue such that a moderate motion or force doesnot cause separation of the material from the tissue on which it isdisposed. The physical bond that is created between the material and thetissue that requires hemostasis may have one or several bases includingelectrostatic bonding and covalent bonding, but any mechanism by whichthe adherence occurs in contemplated herein.

The terms “adhesive” and “adhesivity” similarly refer to the existenceof a physical bond between two materials such as a hemostat of thepresent invention and the tissue to which the hemostat is applied. Anadhesive is a material which adheres to tissue or other material andwhich may be used to constrain the separation of two tissue masses.Adhesivity is the property or degree to which a material adheres to atissue or other material.

As used herein, the term “hydrogel” refers to a material of solid orsemi-solid texture that comprises water. Hydrogels are formed by athree-dimensional network of molecular structures within which water,among other substances, may be retained. The three-dimensional molecularnetwork may be held together by covalent chemical bonds, or by ionicbonds, or by any combination thereof.

The act of “gelation” refers to the formation of a gel, for example, ahydrogel.

A “saccharide” as used herein refers to a carbohydrate. The term“carbohydrate” includes the class of compounds commonly known as sugars,in addition to compounds that are chemically related to sugars. The termthus includes simple “monosaccharide” sugars, “disaccharide” sugars aswell as polymeric “polysaccharides.” The term encompasses a group ofcompounds including sugars, starches, gums, cellulose andhemicelluloses. The term further encompasses sugar derivatives such asamino-sugars, for example, 2-amino-2-deoxyglucose, as well as theiroligomers and polymers; sulfated sugars; and sugars with hydroxyl,amino, carboxyl and other groups.

As used herein, the term “chitosan” refers to a polysaccharide polymer,either obtained from a natural source such as chitin, or syntheticallyprepared. Chemically, chitosan is predominantly a polymer ofβ-1,4-linked 2-amino-2-deoxyglucose monomers. When prepared from anatural source, the usual natural source is chitin, a major constituentof the shells of arthropods (e.g., crabs, lobsters, and shrimp),mollusks, annelids, and cephalopods (e.g., squid). Other natural sourcesof chitin include plants (e.g., fungi and algae). Chitin is chemically apolymer comprising β-1,4-linked 2-acetamino-2-deoxyglucose monomers.After isolation of chitin from its natural source, it is treated tocause hydrolysis of the acetamido group without cleavage of thesugar-sugar bonds, typically through alkaline hydrolysis. Chitosan isnot a single molecular entity, but rather comprises polymeric chains ofvarious lengths.

As used herein, the term “raw chitosan material” refers to chitosanpreparation derived from chitin derived from arthropods (e.g., crabs,lobsters, and shrimp), mollusks, annelids, cephalopods (e.g., squid), orplants (e.g., fungi) that has been treated to cause hydrolysis of theacetamido groups without substantial cleavage of sugar-sugar bonds, forexample, by alkaline hydrolysis.

As used herein, the term “alkylated chitosan” refers to a chitosanmolecule in which a carbon containing moiety has been covalently bonded.The term “alkylated chitosan” comprises a large number of possiblechemical structures that share a common feature that chemical bonds havebeen formed between the components of the chitosan molecules and atleast one carbon atom in each of the molecules that are bonded to thechitosan. For example, alkylated chitosan includes the methylation ofchitosan, in which bonds are formed between methyl radicals or groupsand atoms within the chitosan molecule, such as nitrogen, oxygen orcarbon atoms. Other carbon-containing groups may likewise be chemicallybonded to chitosan molecules to produce an alkylated chitosan. Examplesinclude poly(oxyalkylene)chitosan, wherein poly(oxyethylene), orpolyethyleneglycol, chains are covalently bonded to the chitosanbackbone, as well as acrylated chitosans, formed by alkylation ofchitosan with acrylates.

As used herein, the term “acrylated chitosan” refers to an alkylatedchitosan wherein one or more acrylate groups have been allowed to reactwith, and form chemical bonds to, the chitosan molecule. An acrylate isa molecule containing an 4-unsaturated carbonyl group; thus, acrylicacid is prop-2-enoic acid. The acrylate may bond to the chitosan througha Michael addition of the chitosan nitrogen atoms with the acrylate.

As used herein, the term “degree of substitution” of a polymeric speciesrefers to the ratio of the average number of substituent groups, forexample an alkyl substituent, per monomeric unit of the polymer.

As used herein, the terms “swell”, “swells” or “swelling” refers to theincrease in mass and/or volume of a material (e.g., a hemostatichydrogel composition of the present invention), by greater than about 3%of the material's original mass and/or volume following exposure to aphysiological fluid, body fluid, or aqueous medium.

As used herein, the term “aqueous medium” refers to a liquid mediumcomposed largely, but not necessarily exclusively, of water. Othercomponents may also be present, such as salts, co-solvents, buffers,stabilizers, dispersants, colorants and the like.

As used herein, the terms “subject” and “patient” refer to organisms tobe treated by the methods and/or compositions described herein. Suchorganisms are preferably mammals (e.g., murines, simians, equines,bovines, porcines, canines, felines, and the like), and more preferablyhumans.

As used herein, the term “effective amount” refers to the amount of acompound (e.g., a compound of the present invention) sufficient toeffect beneficial or desired results. An effective amount can beadministered in one or more administrations, applications or dosages andis not intended to be limited to a particular formulation oradministration route. As used herein, the terms “treat,” “treating,” and“treatment” include any effect, e.g., lessening, reducing, modulating,ameliorating or eliminating, that results in the improvement of thecondition, disease, disorder, and the like, or ameliorating a symptomthereof.

The phrase “therapeutically-effective amount” as used herein means thatamount of a compound, material, or composition comprising a compound ofthe present invention which is effective for producing some desiredtherapeutic effect in a subject.

In the application, where an element or component is said to be includedin and/or selected from a list of recited elements or components, itshould be understood that the element or component can be any one of therecited elements or components, or the element or component can beselected from a group consisting of two or more of the recited elementsor components.

Further, it should be understood that elements and/or features of acomposition or a method described herein can be combined in a variety ofways without departing from the spirit and scope of the presentinvention, whether explicit or implicit herein. For example, wherereference is made to a particular compound, that compound can be used invarious embodiments of compositions of the present invention and/or inmethods of the present invention, unless otherwise understood from thecontext. In other words, within this application, embodiments have beendescribed and depicted in a way that enables a clear and conciseapplication to be written and drawn, but it is intended and will beappreciated that embodiments may be variously combined or separatedwithout parting from the present teachings and invention(s). Forexample, it will be appreciated that all features described and depictedherein can be applicable to all aspects of the invention(s) describedand depicted herein.

It should be understood that the expression “at least one of” includesindividually each of the recited objects after the expression and thevarious combinations of two or more of the recited objects unlessotherwise understood from the context and use. The expression “and/or”in connection with three or more recited objects should be understood tohave the same meaning unless otherwise understood from the context.

The use of the term “include,” “includes,” “including,” “have,” “has,”“having,” “contain,” “contains,” or “containing,” including grammaticalequivalents thereof, should be understood generally as open-ended andnon-limiting, for example, not excluding additional unrecited elementsor steps, unless otherwise specifically stated or understood from thecontext.

Where the use of the term “about” is before a quantitative value, thepresent invention also includes the specific quantitative value itself,unless specifically stated otherwise. As used herein, the term “about”refers to a ±10% variation from the nominal value unless otherwiseindicated or inferred.

It should be understood that the order of steps or order for performingcertain actions is immaterial so long as the present invention remainoperable. Moreover, two or more steps or actions may be conductedsimultaneously.

The use of any and all examples, or exemplary language herein, forexample, “such as” or “including,” is intended merely to illustratebetter the present invention and does not pose a limitation on the scopeof the invention unless claimed. No language in the specification shouldbe construed as indicating any non-claimed element as essential to thepractice of the present invention.

Throughout the description, where compositions and kits are described ashaving, including, or comprising specific components, or where processesand methods are described as having, including, or comprising specificsteps, it is contemplated that, additionally, there are compositions andkits of the present invention that consist essentially of, or consistof, the recited components, and that there are processes and methodsaccording to the present invention that consist essentially of, orconsist of, the recited processing steps.

As a general matter, compositions specifying a percentage are by weightunless otherwise specified. Further, if a variable is not accompanied bya definition, then the previous definition of the variable controls.

The following sections describe the synthesis of acrylated chitosan andoxidized dextran which are used to form the hemostatic compositionsdescribed herein.

II Acrylated Chitosan

In one aspect, the invention provides acrylated chitosan useful inproducing the hemostatic compositions described herein. In certainembodiments, the acrylated chitosan compositions comprise acrylatedchitosan comprising:

-   -   (i) from about 0.01 to about 0.3 mole fraction of a first        monomer of formula (I)

-   -   (ii) from about 0.3 to about 0.75 mole fraction of a second        monomer of formula (II)

and

-   -   (iii) from about 0.2 to about 0.7 mole fraction of a third        monomer of formula (III)

The term mole fraction, as used herein, refers to the mole fraction of amonomer present in a polymer and/or hydrogel composition disclosedherein, as determined without including the contribution of solvent(e.g., water) in the polymer and/or hydrogel composition.

In certain embodiments, the acrylated chitosan comprises from about 0.01to about 0.26, from about 0.02 to about 0.26, from about 0.03 to about0.26, from about 0.04 to about 0.26, from about 0.05 to about 0.26, fromabout 0.06 to about 0.26, from about 0.02 to about 0.23, from about 0.02to about 0.19, from about 0.02 to about 0.16, from about 0.02 to about0.13, from about 0.02 to about 0.1, from about 0.02 to about 0.07, fromabout 0.02 to about 0.04, from about 0.03 to about 0.23, from about 0.03to about 0.19, from about 0.03 to about 0.16, from about 0.03 to about0.13, from about 0.03 to about 0.1, from about 0.03 to about 0.07, fromabout 0.03 to about 0.04, from about 0.04 to about 0.23, from about 0.04to about 0.19, from about 0.04 to about 0.16, from about 0.04 to about0.13, from about 0.04 to about 0.1, from about 0.04 to about 0.07, fromabout 0.05 to about 0.23, from about 0.05 to about 0.19, from about 0.05to about 0.16, from about 0.05 to about 0.13, from about 0.05 to about0.1, from about 0.05 to about 0.07, from about 0.06 to about 0.23, fromabout 0.06 to about 0.19, from about 0.06 to about 0.16, from about 0.06to about 0.13, from about 0.06 to about 0.1, or from about 0.06 to about0.07 mole fraction of the first monomer of formula (I). In certainembodiments, the acrylated chitosan comprises from about 0.01 to about0.26 mole fraction of the first monomer of formula (I). In certainembodiments, the acrylated chitosan comprises from about 0.02 to about0.04, about 0.04 to about 0.07, or about 0.06 to about 0.16 molefraction of the first monomer of formula (I).

In certain embodiments, the acrylated chitosan comprises from about 0.35to about 0.65, from about 0.4 to about 0.65, from about 0.45 to about0.65, from about 0.5 to about 0.65, from about 0.55 to about 0.65, fromabout 0.6 to about 0.65, from about 0.35 to about 0.6, from about 0.35to about 0.6, from about 0.35 to about 0.55, from about 0.35 to about0.5, from about 0.35 to about 0.45, from about 0.35 to about 0.4, fromabout 0.4 to about 0.6, from about 0.4 to about 0.55, from about 0.4 toabout 0.5, from about 0.4 to about 0.45, from about 0.45 to about 0.6,from about 0.45 to about 0.55, from about 0.45 to about 0.5, from about0.5 to about 0.6, from about 0.5 to about 0.55, or from about 0.55 toabout 0.6 mole fraction of the second monomer of formula (II). Incertain embodiments, wherein the acrylated chitosan comprises from about0.35 to about 0.65 mole fraction of the second monomer of formula (II).In certain embodiments, wherein the acrylated chitosan comprises fromabout 0.35 to about 0.5, from about 0.45 to about 0.55, from about 0.5to about 0.6 mole fraction of the second monomer of formula (II).

In certain embodiments, the acrylated chitosan comprises from about 0.3to about 0.6, from about 0.35 to about 0.6, from about 0.4 to about 0.6,from about 0.45 to about 0.6, from about 0.5 to about 0.6, from about0.55 to about 0.6, from about 0.3 to about 0.55, from about 0.3 to about0.5, from about 0.3 to about 0.45, from about 0.3 to about 0.4, fromabout 0.3 to about 0.35, from about 0.35 to about 0.55, from about 0.35to about 0.5, from about 0.35 to about 0.45, from about 0.35 to about0.4, from about 0.4 to about 0.55, from about 0.4 to about 0.5, fromabout 0.4 to about 0.45, from about 0.45 to about 0.55, from about 0.45to about 0.5, or from about 0.5 to about 0.55 mole fraction of the thirdmonomer of formula (III). In certain embodiments, wherein the acrylatedchitosan comprises from about 0.3 to about 0.6 mole fraction of thethird monomer of formula (III). In certain embodiments, wherein theacrylated chitosan comprises from about 0.3 to about 0.45, about 0.4 toabout 0.45 mole fraction, or about 0.4 to about 0.55 of the thirdmonomer of formula (III).

In certain embodiments, the acrylated chitosan further comprises lessthan about 0.1 mole fraction of a fourth monomer of formula (IV)

In certain embodiments, the acrylated chitosan further comprises lessthan about 0.03 mole fraction of the fourth monomer of formula (IV). Incertain embodiments, the acrylated chitosan further comprises from about0.01 to about 0.03, from about 0.015 to about 0.03, from about 0.02 toabout 0.03, from about 0.025 to about 0.03, from about 0.01 to about0.025, from about 0.01 to about 0.02, from about 0.01 to about 0.015,from about 0.015 to about 0.025, from about 0.015 to about 0.02, or fromabout 0.02 to about 0.025 mole fraction of the fourth monomer of formula(IV). In certain embodiments, the acrylated chitosan further comprisesfrom about 0.01 to about 0.03 mole fraction of the fourth monomer offormula (IV).

In certain embodiments, the acrylated chitosan has a Mw of from about 25kDa to about 400 kDa, from about 25 kDa to about 300 kDa, from about 25kDa to about 200 kDa, from about 50 kDa to about 400 kDa, from about 50kDa to about 300 kDa, from about 50 kDa to about 200 kDa, from about 115kDa to about 200 kDa, from about 115 kDa to about 175 kDa, from about115 kDa to about 165 kDa, from about 115 kDa to about 155 kDa, fromabout 115 kDa to about 145 kDa, from about 115 kDa to about 135 kDa,from about 115 kDa to about 125 kDa, from about 125 kDa to about 200kDa, from about 125 kDa to about 175 kDa, from about 125 kDa to about165 kDa, from about 125 kDa to about 155 kDa, from about 125 kDa toabout 145 kDa, from about 125 kDa to about 135 kDa, from about 135 kDato about 200 kDa, from about 135 kDa to about 175 kDa, from about 135kDa to about 165 kDa, from about 135 kDa to about 155 kDa, from about135 kDa to about 145 kDa, from about 145 kDa to about 200 kDa, fromabout 145 kDa to about 175 kDa, from about 145 kDa to about 165 kDa,from about 145 kDa to about 155 kDa, from about 155 kDa to about 200kDa, from about 155 kDa to about 175 kDa, from about 155 kDa to about165 kDa, from about 165 kDa to about 200 kDa, from about 165 kDa toabout 175 kDa, or from about 175 kDa to about 200 kDa.

In certain embodiments, the acrylated chitosan has a Mw of from about 25kDa to about 400 kDa. In certain embodiments, the acrylated chitosan hasa Mw of from about 115 kDa to about 200 kDa. In certain embodiments, theacrylated chitosan has a Mw of from about 115 kDa to about 165 kDa, fromabout 125 kDa to about 155 kDa, or from about 135 kDa to about 175 kDa.

In certain embodiments, the acrylated chitosan has a Mn of from about 14kDa to about 200 kDa, from about 14 kDa to about 150 kDa, from about 25kDa to about 200 kDa, from about 14 kDa to about 150 kDa, from about 55kDa to about 140 kDa, from about 55 kDa to about 120 kDa, from about 55kDa to about 100 kDa, from about 55 kDa to about 90 kDa, from about 55kDa to about 80 kDa, from about 55 kDa to about 75 kDa, from about 55kDa to about 70 kDa, from about 55 kDa to about 65 kDa, from about 55kDa to about 60 kDa, from about 60 kDa to about 140 kDa, from about 60kDa to about 120 kDa, from about 60 kDa to about 100 kDa, from about 60kDa to about 90 kDa, from about 60 kDa to about 80 kDa, from about 60kDa to about 75 kDa, from about 60 kDa to about 70 kDa, from about 60kDa to about 65 kDa, from about 65 kDa to about 140 kDa, from about 65kDa to about 120 kDa, from about 65 kDa to about 100 kDa, from about 65kDa to about 90 kDa, from about 65 kDa to about 80 kDa, from about 65kDa to about 75 kDa, from about 65 kDa to about 70 kDa, from about 70kDa to about 140 kDa, from about 70 kDa to about 120 kDa, from about 70kDa to about 100 kDa, from about 70 kDa to about 90 kDa, from about 70kDa to about 80 kDa, from about 70 kDa to about 75 kDa, from about 75kDa to about 140 kDa, from about 75 kDa to about 120 kDa, from about 75kDa to about 100 kDa, from about 75 kDa to about 90 kDa, from about 75kDa to about 80 kDa, from about 80 kDa to about 140 kDa, from about 80kDa to about 120 kDa, from about 80 kDa to about 100 kDa, from about 80kDa to about 90 kDa, from about 90 kDa to about 140 kDa, from about 90kDa to about 120 kDa, from about 90 kDa to about 100 kDa, from about 100kDa to about 140 kDa, from about 100 kDa to about 120 kDa, or from about120 kDa to about 140 kDa.

In certain embodiments, wherein the acrylated chitosan has a Mn of fromabout 14 kDa to about 200 kDa. In certain embodiments, wherein theacrylated chitosan has a Mn of from about 55 kDa to about 140 kDa. Incertain embodiments, wherein the acrylated chitosan has a Mn of fromabout 55 kDa to about 75 kDa, from about 65 kDa to about 75 kDa, or fromabout 65 kDa to about 100 kDa.

In certain embodiments, the acrylated chitosan has a PDI of from about1.5 (Mw/Mn) to about 3.5 (Mw/Mn), from about 1.7 (Mw/Mn) to about 3.5(Mw/Mn), from about 2.1 (Mw/Mn) to about 3.5 (Mw/Mn), from about 2.4(Mw/Mn) to about 3.5 (Mw/Mn), from about 2.7 (Mw/Mn) to about 3.5(Mw/Mn), from about 3.0 (Mw/Mn) to about 3.5 (Mw/Mn), from about 3.3(Mw/Mn) to about 3.5 (Mw/Mn), from about 1.5 (Mw/Mn) to about 3.3(Mw/Mn), from about 1.5 (Mw/Mn) to about 3.0 (Mw/Mn), from about 1.5(Mw/Mn) to about 2.7 (Mw/Mn), from about 1.5 (Mw/Mn) to about 2.4(Mw/Mn), from about 1.5 (Mw/Mn) to about 2.1 (Mw/Mn), from about 1.5(Mw/Mn) to about 1.8 (Mw/Mn), from about 1.7 (Mw/Mn) to about 3.3(Mw/Mn), from about 1.7 (Mw/Mn) to about 3.0 (Mw/Mn), from about 1.7(Mw/Mn) to about 2.7 (Mw/Mn), from about 1.7 (Mw/Mn) to about 2.4(Mw/Mn), from about 1.7 (Mw/Mn) to about 2.1 (Mw/Mn), from about 1.9(Mw/Mn) to about 3.3 (Mw/Mn), from about 1.9 (Mw/Mn) to about 3.0(Mw/Mn), from about 1.9 (Mw/Mn) to about 2.7 (Mw/Mn), from about 1.9(Mw/Mn) to about 2.4 (Mw/Mn), from about 1.9 (Mw/Mn) to about 2.1(Mw/Mn), from about 2.1 (Mw/Mn) to about 3.3 (Mw/Mn), from about 2.1(Mw/Mn) to about 3.0 (Mw/Mn), from about 2.1 (Mw/Mn) to about 2.7(Mw/Mn), from about 2.1 (Mw/Mn) to about 2.4 (Mw/Mn), from about 2.3(Mw/Mn) to about 3.3 (Mw/Mn), from about 2.3 (Mw/Mn) to about 3.0(Mw/Mn), from about 2.3 (Mw/Mn) to about 2.7 (Mw/Mn), from about 2.3(Mw/Mn) to about 2.4 (Mw/Mn), from about 2.5 (Mw/Mn) to about 3.3(Mw/Mn), from about 2.5 (Mw/Mn) to about 3.0 (Mw/Mn), from about 2.5(Mw/Mn) to about 2.7 (Mw/Mn), from about 2.7 (Mw/Mn) to about 3.3(Mw/Mn), from about 2.7 (Mw/Mn) to about 3.0 (Mw/Mn), from about 2.9(Mw/Mn) to about 3.3 (Mw/Mn), from about 2.9 (Mw/Mn) to about 3.0(Mw/Mn), or from about 3.1 (Mw/Mn) to about 3.3 (Mw/Mn).

In certain embodiments, the acrylated chitosan has a PDI of from about1.5 (Mw/Mn) to about 3.5 (Mw/Mn). In certain embodiments, the acrylatedchitosan has a PDI of from about 1.5 (Mw/Mn) to about 2.4 (Mw/Mn), fromabout 1.7 (Mw/Mn) to about 2.1 (Mw/Mn), or from about 2.1 (Mw/Mn) toabout 3.0 (Mw/Mn).

In certain embodiments, the acrylated chitosan has a Mz of from about 80kDa to about 1,200 kDa, from about 80 kDa to about 600 kDa, from about80 kDa to about 370 kDa, from about 150 kDa to about 1,200 kDa, fromabout 150 kDa to about 600 kDa, from about 150 kDa to about 370 kDa,from about 200 kDa to about 370 kDa, from about 200 kDa to about 350kDa, from about 200 kDa to about 320 kDa, from about 200 kDa to about300 kDa, from about 200 kDa to about 270 kDa, from about 200 kDa toabout 260 kDa, from about 200 kDa to about 250 kDa, from about 200 kDato about 240 kDa, from about 200 kDa to about 230 kDa, from about 200kDa to about 220 kDa, from about 200 kDa to about 210 kDa, from about210 kDa to about 370 kDa, from about 210 kDa to about 350 kDa, fromabout 210 kDa to about 320 kDa, from about 210 kDa to about 300 kDa,from about 210 kDa to about 270 kDa, from about 210 kDa to about 260kDa, from about 210 kDa to about 250 kDa, from about 210 kDa to about240 kDa, from about 210 kDa to about 230 kDa, from about 210 kDa toabout 220 kDa, from about 220 kDa to about 370 kDa, from about 220 kDato about 350 kDa, from about 220 kDa to about 320 kDa, from about 220kDa to about 300 kDa, from about 220 kDa to about 270 kDa, from about220 kDa to about 260 kDa, from about 220 kDa to about 250 kDa, fromabout 220 kDa to about 240 kDa, from about 220 kDa to about 230 kDa,from about 230 kDa to about 370 kDa, from about 230 kDa to about 350kDa, from about 230 kDa to about 320 kDa, from about 230 kDa to about300 kDa, from about 230 kDa to about 270 kDa, from about 230 kDa toabout 260 kDa, from about 230 kDa to about 250 kDa, from about 230 kDato about 240 kDa, from about 240 kDa to about 370 kDa, from about 240kDa to about 350 kDa, from about 240 kDa to about 320 kDa, from about240 kDa to about 300 kDa, from about 240 kDa to about 270 kDa, fromabout 240 kDa to about 260 kDa, from about 240 kDa to about 250 kDa,from about 250 kDa to about 370 kDa, from about 250 kDa to about 350kDa, from about 250 kDa to about 320 kDa, from about 250 kDa to about300 kDa, from about 250 kDa to about 270 kDa, from about 250 kDa toabout 260 kDa, from about 260 kDa to about 370 kDa, from about 260 kDato about 350 kDa, from about 260 kDa to about 320 kDa, from about 260kDa to about 300 kDa, from about 260 kDa to about 270 kDa, from about270 kDa to about 370 kDa, from about 270 kDa to about 350 kDa, fromabout 270 kDa to about 320 kDa, from about 270 kDa to about 300 kDa,from about 300 kDa to about 370 kDa, from about 300 kDa to about 350kDa, from about 300 kDa to about 320 kDa, from about 320 kDa to about370 kDa, from about 320 kDa to about 350 kDa, or from about 350 kDa toabout 370 kDa.

In certain embodiments, the acrylated chitosan has a Mz of from about 80kDa to about 1,200 kDa. In certain embodiments, the acrylated chitosanhas a Mz of from about 200 kDa to about 350 kDa. In certain embodiments,the acrylated chitosan has a Mz of from about 200 kDa to about 300 kDa,from about 210 kDa to about 370 kDa, or from about 250 kDa to about 370kDa.

In certain embodiments, the acrylated chitosan has a PDI of from about1.5 (Mz/Mw) to about 7.0 (Mz/Mw), from about 1.5 (Mz/Mw) to about 5.0(Mz/Mw), from about 1.5 (Mz/Mw) to about 3.0 (Mz/Mw), from about 1.6(Mz/Mw) to about 3.0 (Mz/Mw), from about 1.6 (Mz/Mw) to about 2.5(Mz/Mw), from about 1.6 (Mz/Mw) to about 2.0 (Mz/Mw), from about 1.6(Mz/Mw) to about 1.9 (Mz/Mw), from about 1.6 (Mz/Mw) to about 1.8(Mz/Mw), from about 1.6 (Mz/Mw) to about 1.7 (Mz/Mw), from about 1.7(Mz/Mw) to about 3.0 (Mz/Mw), from about 1.7 (Mz/Mw) to about 2.5(Mz/Mw), from about 1.7 (Mz/Mw) to about 2.0 (Mz/Mw), from about 1.7(Mz/Mw) to about 1.9 (Mz/Mw), from about 1.7 (Mz/Mw) to about 1.8(Mz/Mw), from about 1.8 (Mz/Mw) to about 3.0 (Mz/Mw), from about 1.8(Mz/Mw) to about 2.5 (Mz/Mw), from about 1.8 (Mz/Mw) to about 2.0(Mz/Mw), from about 1.8 (Mz/Mw) to about 1.9 (Mz/Mw), from about 1.9(Mz/Mw) to about 3.0 (Mz/Mw), from about 1.9 (Mz/Mw) to about 2.5(Mz/Mw), from about 1.9 (Mz/Mw) to about 2.0 (Mz/Mw), from about 2.0(Mz/Mw) to about 3.0 (Mz/Mw), from about 2.0 (Mz/Mw) to about 2.5(Mz/Mw), or from about 2.5 (Mz/Mw) to about 3.0 (Mz/Mw).

In certain embodiments, the acrylated chitosan has a PDI of from about1.5 (Mz/Mw) to about 7.0 (Mz/Mw). In certain embodiments, the acrylatedchitosan has a PDI of from about 1.6 (Mz/Mw) to about 3.0 (Mz/Mw). Incertain embodiments, the acrylated chitosan has a PDI of from about 1.5(Mz/Mw) to about 2.0 (Mz/Mw), from about 1.6 (Mz/Mw) to about 2.5(Mz/Mw), from about 1.9 (Mz/Mw) to about 3.0 (Mz/Mw).

In certain embodiments, the acrylated chitosan composition comprisesabout 1% (w/w), about 2% (w/w), about 3% (w/w), about 4% (w/w), about 5%(w/w), about 6% (w/w), about 7% (w/w), about 8% (w/w), about 9% (w/w),about 10% (w/w), about 11% (w/w), about 12% (w/w), about 13% (w/w),about 14% (w/w), about 15% (w/w), about 16% (w/w), about 17% (w/w),about 18% (w/w), about 19% (w/w), about 20% (w/w), about 21% (w/w),about 22% (w/w), about 23% (w/w), about 24% (w/w), or about 25% (w/w) ofthe acrylated chitosan.

In certain embodiments, the acrylated chitosan composition comprisesfrom about 1% (w/w) to about 25% (w/w), from about 1% (w/w) to about 20%(w/w), from about 1% (w/w) to about 15% (w/w), from about 1% (w/w) toabout 10% (w/w), from about 1% (w/w) to about 9% (w/w), from about 1%(w/w) to about 8% (w/w), from about 1% (w/w) to about 7% (w/w), fromabout 1% (w/w) to about 6% (w/w), from about 1% (w/w) to about 5% (w/w),from about 1% (w/w) to about 4% (w/w), from about 1% (w/w) to about 3%(w/w), from about 1% (w/w) to about 2% (w/w), from about 2% (w/w) toabout 10% (w/w), from about 2% (w/w) to about 9% (w/w), from about 2%(w/w) to about 8% (w/w), from about 2% (w/w) to about 7% (w/w), fromabout 2% (w/w) to about 6% (w/w), from about 2% (w/w) to about 5% (w/w),from about 2% (w/w) to about 4% (w/w), from about 2% (w/w) to about 3%(w/w), from about 3% (w/w) to about 10% (w/w), from about 3% (w/w) toabout 9% (w/w), from about 3% (w/w) to about 8% (w/w), from about 3%(w/w) to about 7% (w/w), from about 3% (w/w) to about 6% (w/w), fromabout 3% (w/w) to about 5% (w/w), from about 3% (w/w) to about 4% (w/w),from about 4% (w/w) to about 10% (w/w), from about 4% (w/w) to about 9%(w/w), from about 4% (w/w) to about 8% (w/w), from about 4% (w/w) toabout 7% (w/w), from about 4% (w/w) to about 6% (w/w), from about 4%(w/w) to about 5% (w/w), from about 5% (w/w) to about 10% (w/w), fromabout 5% (w/w) to about 9% (w/w), from about 5% (w/w) to about 8% (w/w),from about 5% (w/w) to about 7% (w/w), from about 5% (w/w) to about 6%(w/w), from about 6% (w/w) to about 10% (w/w), from about 6% (w/w) toabout 9% (w/w), from about 6% (w/w) to about 8% (w/w), from about 6%(w/w) to about 7% (w/w), from about 7% (w/w) to about 10% (w/w), fromabout 7% (w/w) to about 9% (w/w), from about 7% (w/w) to about 8% (w/w),from about 8% (w/w) to about 10% (w/w), from about 8% (w/w) to about 9%(w/w), or from about 9% (w/w) to about 10% (w/w) of the acrylatedchitosan. In certain embodiments, the acrylated chitosan compositioncomprises from about 1% (w/w) to about 25% (w/w). In certainembodiments, the acrylated chitosan composition comprises from about 1%(w/w) to about 10% (w/w) of the acrylated chitosan. In certainembodiments, the acrylated chitosan composition comprises from about 2%(w/w) to about 5% (w/w), from about 4% (w/w) to about 7% (w/w), or fromabout 6% (w/w) to about 9% (w/w).

In certain embodiments, the acrylated chitosan composition has aviscosity of from about 10 cP to about 50,000 cP, from about 10 cP toabout 35,000 cP, from about 10 cP to about 25,000 cP, from about 10 cPto about 20,000 cP, from about 10 cP to about 10,000 cP, from about 10cP to about 8,000 cP, from about 10 cP to about 4,000 cP, from about 10cP to about 1,000 cP, from about 10 cP to about 100 cP, from about 100cP to about 25,000 cP, from about 100 cP to about 20,000 cP, from about100 cP to about 10,000 cP, from about 100 cP to about 8,000 cP, fromabout 100 cP to about 4,000 cP, from about 100 cP to about 1,000 cP,from about 1,000 cP to about 25,000 cP, from about 1,000 cP to about20,000 cP, from about 1,000 cP to about 10,000 cP, from about 1,000 cPto about 8,000 cP, from about 1,000 cP to about 4,000 cP, from about4,000 cP to about 25,000 cP, from about 4,000 cP to about 20,000 cP,from about 4,000 cP to about 10,000 cP, from about 4,000 cP to about8,000 cP, from about 8,000 cP to about 25,000 cP, from about 8,000 cP toabout 20,000 cP, from about 8,000 cP to about 10,000 cP, or from about10,000 cP to about 20,000 cP.

In certain embodiments, the acrylated chitosan composition has aviscosity of from about 10 cP to about 50,000 cP. In certainembodiments, the acrylated chitosan composition has a viscosity of fromabout 100 cP to about 25,000 cP. In certain embodiments, the acrylatedchitosan composition has a viscosity of from about 1,000 cP to about10,000 cP, about 8,000 cP to about 20,000 cP, or from about 10,000 cP toabout 25,000 cP.

In certain embodiments, the acrylated chitosan composition has a pH offrom about 5.5 to about 9.0, from about 6.0 to about 9.0, from about 6.5to about 9.0, from about 7.0 to about 9.0, from about 7.5 to about 9.0,from about 8.0 to about 9.0, from about 8.5 to about 9.0, from about 8.0to about 8.8, from about 8.0 to about 8.6, from about 8.0 to about 8.4,from about 8.0 to about 8.2, from about 8.2 to about 8.8, from about 8.2to about 8.6, from about 8.2 to about 8.4, from about 8.4 to about 8.8,from about 8.4 to about 8.6, or from about 8.6 to about 8.8. In certainembodiments, the acrylated chitosan composition has a pH of from about5.5 to about 9.0. In certain embodiments, the acrylated chitosancomposition has a pH of from about 8.0 to about 8.4, from about 8.2 toabout 8.8, or from about 8.2 to about 8.8.

In certain embodiments, the acrylated chitosan composition has aconductivity of from about 0.75 (mS/cm) to about 25.0 (mS/cm), fromabout 0.75 (mS/cm) to about 20.0 (mS/cm), from about 0.75 (mS/cm) toabout 15.0 (mS/cm), from about 0.75 (mS/cm) to about 10.0 (mS/cm), fromabout 0.75 (mS/cm) to about 5.0 (mS/cm), from about 0.75 (mS/cm) toabout 4.0 (mS/cm), from about 0.75 (mS/cm) to about 3.0 (mS/cm), fromabout 0.75 (mS/cm) to about 2.0 (mS/cm), from about 0.75 (mS/cm) toabout 1.0 (mS/cm), from about 1.0 (mS/cm) to about 10.0 (mS/cm), fromabout 1.0 (mS/cm) to about 5.0 (mS/cm), from about 1.0 (mS/cm) to about4.0 (mS/cm), from about 1.0 (mS/cm) to about 3.0 (mS/cm), from about 1.0(mS/cm) to about 2.0 (mS/cm), from about 2.0 (mS/cm) to about 10.0(mS/cm), from about 2.0 (mS/cm) to about 5.0 (mS/cm), from about 2.0(mS/cm) to about 4.0 (mS/cm), from about 2.0 (mS/cm) to about 3.0(mS/cm), from about 3.0 (mS/cm) to about 10.0 (mS/cm), from about 3.0(mS/cm) to about 5.0 (mS/cm), from about 3.0 (mS/cm) to about 4.0(mS/cm), from about 4.0 (mS/cm) to about 10.0 (mS/cm), from about 4.0(mS/cm) to about 5.0 (mS/cm), or from about 5.0 (mS/cm) to about 10.0(mS/cm).

In certain embodiments, the acrylated chitosan composition has aconductivity of from about 0.75 (mS/cm) to about 25.0 (mS/cm). Incertain embodiments, the acrylated chitosan composition has aconductivity of from about 0.75 (mS/cm) to about 10 (mS/cm). In certainembodiments, the acrylated chitosan composition has a conductivity offrom about 1.0 (mS/cm) to about 5.0 (mS/cm), from about 3.0 (mS/cm) toabout 10.0 (mS/cm), or from about 5.0 (mS/cm) to about 10.0 (mS/cm).

In certain embodiments, the acrylated chitosan has two or moreproperties selected from a Mw of from about 25 kDa to about 400 kDa, aMn of from about 14 kDa to about 200 kDa, a PDI of from about 1.5 toabout 3.5 (Mw/Mn), an Mz of from about 80 kDa to about 1,200 kDa, and aPDI of from about 1.5 to about 7.0 (Mz/Mw).

In certain embodiments, the acrylated chitosan composition comprises aplurality (e.g., 2, 3, 4, 5, 6, 7, or 8) or all of the followingfeatures: (i) a Mw of from about 115 kDa to about 200 kDa, (ii) a Mn offrom about 55 kDa to about 140 kDa, (iii) a Mz of from about 200 kDa toabout 350 kDa, (iv) a PDI (Mw/Mn) of from about 1.5 to about 3.5, (v) aPDI (Mz/Mw) of from about 1.6 to about 3.0, (vi) a mole fraction of themonomer of formula (I) of from about 0.011 to about 0.26, (vii) a molefraction of the monomer of formula (II) of from about 0.35 to about0.65, (viii) a mole fraction of the monomer of formula (III) of fromabout 0.32 to about 0.6, and (ix) a mole fraction of the monomer offormula (IV) of from about 0.09 to about 0.028.

In certain embodiments, the acrylated chitosan composition comprises aplurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) or all of thefollowing features: (i) a Mw of from about 115 kDa to about 200 kDa,(ii) a Mn of from about 55 kDa to about 140 kDa, (iii) a Mz of fromabout 200 kDa to about 350 kDa, (iv) a PDI (Mw/Mn) of from about 1.5 toabout 3.5, (v) a PDI (Mz/Mw) of from about 1.6 to about 3.0, (vi) a molefraction of the monomer of formula (I) of from about 0.011 to about0.26, (vii) a mole fraction of the monomer of formula (II) of from about0.35 to about 0.65, (viii) a mole fraction of the monomer of formula(III) of from about 0.32 to about 0.6, (ix) a mole fraction of themonomer of formula (IV) of from about 0.09 to about 0.028, (x) aconcentration of the acrylated chitosan in an aqueous medium or fromabout 1% (w/w) to about 10% (w/w), (xi) a viscosity of from about 10 cPto about 20,300 cP, (xii) a pH of from about 5.5 to about 9.0, and(xiii) a conductivity of from about 0.75 mS/cm to about 10.0 mS/cm.

III Methods of Making Acrylated Chitosan

In one aspect, the invention provides a method of preparing an acrylatedchitosan composition useful in making a hemostatic composition describedherein. The method comprises the steps of:

-   -   (a) contacting a raw chitosan material with an acetic acid        solution to form a chitosan intermediate;    -   (b) contacting the intermediate chitosan material with acrylic        acid to form an acrylated chitosan intermediate; and    -   (c) purifying the acrylated chitosan intermediate to produce an        acrylated chitosan composition of the present invention.

In certain embodiments, during step (a), the raw chitosan material iscontacted with the acetic acid solution at a pressure of about 0.5atmospheres, about 1.0 atmosphere, about 1.5 atmospheres, about 2atmospheres, about 2.5 atmospheres, about 3.0 atmospheres, about 3.5atmospheres, about 4.0 atmospheres, about 4.5 atmospheres, or about 5.0atmospheres. In certain embodiments, during step (a), the raw chitosanmaterial is contacted with the acetic acid solution at a pressure ofabout 2 atmospheres.

In certain embodiments, during step (a), the raw chitosan material iscontacted with the acetic acid solution at a temperature of about 80°C., about 85° C., about 90° C., about 95° C., about 100° C., about 115°C., or about 120° C. In certain embodiments, during step (a), the rawchitosan material is contacted with the acetic acid solution at atemperature from about 80° C. to about 120° C., from about 90° C. toabout 120° C., from about 100° C. to about 120° C., from about 110° C.to about 120° C., from about 80° C. to about 110° C., from about 80° C.to about 100° C., from about 80° C. to about 90° C., from about 90° C.to about 110° C., from about 90° C. to about 100° C., or from about 100°C. to about 110° C. In certain embodiments, during step (a), the rawchitosan material is contacted with the acetic acid solution at atemperature from about 80° C. to about 120° C.

In certain embodiments, during step (a), the acetic acid solution is a1% (v/v) acetic acid solution, a 2% (v/v) acetic acid solution, a 3%(v/v) acetic acid solution, a 4% (v/v) acetic acid solution, a 5% (v/v)acetic acid solution, a 6% (v/v) acetic acid solution, a 7% (v/v) aceticacid solution, a 8% (v/v) acetic acid solution, a 9% (v/v) acetic acidsolution, or a 10% (v/v) acetic acid solution. In certain embodiments,during step (a), the acetic acid solution is a 1% (v/v) acetic acidsolution.

In certain embodiments, during step (a), the acetic acid solution is a1% (w/v) acetic acid solution, a 2% (w/v) acetic acid solution, a 3%(w/v) acetic acid solution, a 4% (w/v) acetic acid solution, a 5% (w/v)acetic acid solution, a 6% (w/v) acetic acid solution, a 7% (w/v) aceticacid solution, a 8% (w/v) acetic acid solution, a 9% (w/v) acetic acidsolution, or a 10% (w/v) acetic acid solution. In certain embodiments,during step (a), the acetic acid solution is a 1% (w/v) acetic acidsolution.

In certain embodiments, during step (a), the raw chitosan material has aweight-average molecular weight (Mw) of from about 40 kDa to about 1,000kDa, about 50 kDa to about 750 kDa, about 50 kDa to about 500 kDa, about100 kDa to about 750 kDa, from about 100 kDa to about 500 kDa, fromabout 100 kDa to about 450 kDa, from about 100 kDa to about 400 kDa,from about 100 kDa to about 350 kDa, from about 100 kDa to about 300kDa, from about 100 kDa to about 250 kDa, from about 100 kDa to about200 kDa, from about 100 kDa to about 150 kDa, from about 150 kDa toabout 500 kDa, from about 150 kDa to about 450 kDa, from about 150 kDato about 400 kDa, from about 150 kDa to about 350 kDa, from about 150kDa to about 300 kDa, from about 150 kDa to about 250 kDa, from about150 kDa to about 200 kDa, from about 200 kDa to about 500 kDa, fromabout 200 kDa to about 450 kDa, from about 200 kDa to about 400 kDa,from about 200 kDa to about 350 kDa, from about 200 kDa to about 300kDa, from about 200 kDa to about 250 kDa, from about 250 kDa to about500 kDa, from about 250 kDa to about 450 kDa, from about 250 kDa toabout 400 kDa, from about 250 kDa to about 350 kDa, from about 250 kDato about 300 kDa, from about 300 kDa to about 500 kDa, from about 300kDa to about 450 kDa, from about 300 kDa to about 400 kDa, from about300 kDa to about 350 kDa, from about 350 kDa to about 500 kDa, fromabout 350 kDa to about 450 kDa, from about 350 kDa to about 400 kDa,from about 400 kDa to about 500 kDa, from about 400 kDa to about 450kDa, or from about 450 kDa to about 500 kDa. In certain embodiments,during step (a), the raw chitosan material has a Mw of from about 40 kDato about 1,000 kDa. In certain embodiments, during step (a), the rawchitosan material has a Mw of from about 100 kDa to about 500 kDa. Incertain embodiments, during step (a), the raw chitosan material has a Mwof from about 100 kDa to about 250 kDa, from about 200 kDa to about 350kDa, or from about 300 kDa to about 450 kDa.

In certain embodiments, during step (a), the raw chitosan material has anumber-average molecular weight (Mn) of greater than 10 kDa, greaterthan 15 kDa, greater than 20 kDa, greater than 25 kDa, greater than 30kDa, greater than 35 kDa, greater than 40 kDa, greater than 45 kDa,greater than 50 kDa, greater than 55 kDa, greater than 60 kDa, greaterthan 65 kDa, greater than 70 kDa, greater than 75 kDa, greater than 80kDa, greater than 85 kDa, greater than 90 kDa, greater than 95 kDa,greater than 100 kDa, greater than 105 kDa, greater than 110 kDa,greater than 115 kDa, greater than 120 kDa. In certain embodiments,during step (a), the raw chitosan material has a number-averagemolecular weight (Mn) of greater than 20 kDa. In certain embodiments,during step (a), the raw chitosan material has a number-averagemolecular weight (Mn) of greater than 60 kDa. In certain embodiments,during step (a), the raw chitosan material has a number-averagemolecular weight (Mn) in the range of from about 20 kDa to about 650kDa, from about 20 kDa to about 400 kDa, from about 20 kDa to about 300kDa, from about 20 kDa to about 200 kDa, from about 40 kDa to about 400kDa, from about 40 kDa to about 300 kDa, from about 40 kDa to about 200kDa, from about 40 kDa to about 100 kDa, from about 100 kDa to about 300kDa, from about 100 kDa to about 200 kDa, or from about 200 kDa to about300 kDa. In certain embodiments, during step (a), the raw chitosanmaterial has a number-average molecular weight (Mn) in the range of fromabout 20 kDa to about 650 kDa. In certain embodiments, during step (a),the raw chitosan material has a number-average molecular weight (Mn) inthe range of from about 40 kDa to about 300 kDa.

In certain embodiments, during step (a), the raw chitosan material has apolydispersity index (PDI) of from about 1.5 (Mw/Mn) to about 5.0(Mw/Mn), from about 1.6 (Mw/Mn) to about 4.5 (Mw/Mn), from about 1.6(Mw/Mn) to about 4.0 (Mw/Mn), from about 1.7 (Mw/Mn) to about 4.0(Mw/Mn), from about 1.7 (Mw/Mn) to about 3.5 (Mw/Mn), from about 1.7(Mw/Mn) to about 3.0 (Mw/Mn), from about 1.7 (Mw/Mn) to about 2.5(Mw/Mn), from about 1.7 (Mw/Mn) to about 2.0 (Mw/Mn), from about 1.8(Mw/Mn) to about 4.0 (Mw/Mn), from about 1.8 (Mw/Mn) to about 3.5(Mw/Mn), from about 1.8 (Mw/Mn) to about 3.0 (Mw/Mn), from about 1.8(Mw/Mn) to about 2.5 (Mw/Mn), from about 1.8 (Mw/Mn) to about 2.0(Mw/Mn), from about 1.9 (Mw/Mn) to about 4.0 (Mw/Mn), from about 1.9(Mw/Mn) to about 3.5 (Mw/Mn), from about 1.9 (Mw/Mn) to about 3.0(Mw/Mn), from about 1.9 (Mw/Mn) to about 2.5 (Mw/Mn), from about 1.9(Mw/Mn) to about 2.0 (Mw/Mn), from about 2.0 (Mw/Mn) to about 4.0(Mw/Mn), from about 2.0 (Mw/Mn) to about 3.5 (Mw/Mn), from about 2.0(Mw/Mn) to about 3.0 (Mw/Mn), or from about 2.0 (Mw/Mn) to about 2.5(Mw/Mn). In certain embodiments, during step (a), the raw chitosanmaterial has a polydispersity index of from about 1.5 (Mw/Mn) to about5.0 (Mw/Mn). In certain embodiments, during step (a), the raw chitosanmaterial has a polydispersity index of from about 1.7 (Mw/Mn) to about4.0 (Mw/Mn). In certain embodiments, during step (a), the raw chitosanmaterial has a polydispersity index of from about 1.6 (Mw/Mn) to about3.0 (Mw/Mn), about 1.8 (Mw/Mn) to about 3.5 (Mw/Mn) or from about 2.0(Mw/Mn) to about 4.0 (Mw/Mn).

In certain embodiments, during step (a), the raw chitosan material has az-average molecular weight (Mz) of from about 100 kDa to about 2000 kDa,about 100 kDa to about 1500 kDa, about 100 kDa to about 1000 kDa, about200 kDa to about 2000 kDa, about 100 kDa to about 15000 kDa, about 100kDa to about 1000 kDa, from about 250 kDa to about 750 kDa, from about250 kDa to about 600 kDa, from about 250 kDa to about 500 kDa, fromabout 250 kDa to about 400 kDa, from about 250 kDa to about 300 kDa,from about 300 kDa to about 750 kDa, from about 300 kDa to about 600kDa, from about 300 kDa to about 500 kDa, from about 300 kDa to about400 kDa, from about 300 kDa to about 350 kDa, from about 350 kDa toabout 750 kDa, from about 350 kDa to about 600 kDa, from about 350 kDato about 500 kDa, from about 350 kDa to about 400 kDa, from about 400kDa to about 750 kDa, from about 400 kDa to about 600 kDa, from about400 kDa to about 500 kDa, from about 500 kDa to about 750 kDa, fromabout 500 kDa to about 600 kDa, or from about 600 kDa to about 750 kDa.In certain embodiments, during step (a), the raw chitosan material has aMz of from about 100 kDa to about 2000 kDa. In certain embodiments,during step (a), the raw chitosan material has a Mz of from about 250kDa to about 750 kDa. In certain embodiments, during step (a), the rawchitosan material has a Mz of from about 250 kDa to about 500 kDa, fromabout 350 kDa to about 600 kDa or from about 400 kDa to about 750 kDa.

In certain embodiments, during step (a), the raw chitosan material has apolydispersity index (PDI) of from about 1.5 (Mz/Mw) to about 4.0(Mz/Mw), from about 1.5 (Mz/Mw) to about 3.75 (Mz/Mw), from about 1.5(Mz/Mw) to about 3.5 (Mz/Mw), from about 1.5 (Mz/Mw) to about 3.0(Mz/Mw), from about 1.5 (Mz/Mw) to about 2.5 (Mz/Mw), from about 1.5(Mz/Mw) to about 2.0 (Mz/Mw), from about 2.0 (Mz/Mw) to about 3.75(Mz/Mw), from about 2.0 (Mz/Mw) to about 3.5 (Mz/Mw), from about 2.0(Mz/Mw) to about 3.0 (Mz/Mw), from about 2.0 (Mz/Mw) to about 2.5(Mz/Mw), from about 2.5 (Mz/Mw) to about 3.75 (Mz/Mw), from about 2.5(Mz/Mw) to about 3.5 (Mz/Mw), from about 2.5 (Mz/Mw) to about 3.0(Mz/Mw), from about 3.0 (Mz/Mw) to about 3.75 (Mz/Mw) or from about 3.0(Mz/Mw) to about 3.5 (Mz/Mw). In certain embodiments, during step (a),the raw chitosan material has a polydispersity index (PDI) of from about1.5 (Mz/Mw) to about 4.0 (Mz/Mw). In certain embodiments, during step(a), the raw chitosan material has a polydispersity index (PDI) of fromabout 1.5 (Mz/Mw) to about 3.5 (Mz/Mw). In certain embodiments, duringstep (a), the raw chitosan material has a polydispersity index (PDI) offrom about 1.5 (Mz/Mw) to about 2.0 (Mz/Mw), from about 1.5 (Mz/Mw) toabout 3.0 (Mz/Mw), or from about 2.5 (Mz/Mw) to about 3.75 (Mz/Mw).

In certain embodiments, during step (a), the PDI (Mw/Mn) of the chitosanintermediate is from about 10% to about 60%, from about 10% to about50%, from about 10% to about 40%, from about 15% to about 40%, fromabout 20% to about 40%, from about 30% to about 40%, from about 15% toabout 30%, from about 15% to about 20%, or from about 20% to about 30%less than the PDI (Mw/Mn) of the raw chitosan material. In certainembodiments, during step (a), the PDI (Mw/Mn) of the chitosanintermediate is from about 10% to about 60% less than the PDI (Mw/Mn) ofthe raw chitosan material. In certain embodiments, during step (a), thePDI (Mw/Mn) of the chitosan intermediate is from about 15% to about 40%less than the PDI (Mw/Mn) of the raw chitosan material.

In certain embodiments, during step (a), the raw chitosan material has adegree of deacetylation of from about 65% to about 100%, from about 70%to about 100%, from about 75% to about 100%, from about 80% to about100%, from about 85% to about 100%, from about 90% to about 100%, fromabout 95% to about 100%, about 65% to about 99%, from about 70% to about99%, from about 72.5% to about 99%, from about 75% to about 99%, fromabout 77.5% to about 99%, from about 80.0% to about 99%, about 65% toabout 98%, from about 70% to about 98%, from about 72.5% to about 98%,from about 75% to about 98%, from about 77.5% to about 98%, from about80.0% to about 98%, about 65% to about 97%, from about 70% to about 97%,from about 72.5% to about 97%, from about 75% to about 97%, from about77.5% to about 97%, or from about 80.0% to about 97%. In certainembodiments, during step (a), the raw chitosan material has a degree ofdeacetylation of from about 65% to about 100%. In certain embodiments,during step (a), the raw chitosan material has a degree of deacetylationof from about 70% to about 98%. In certain embodiments, during step (a),the raw chitosan material has a degree of deacetylation of from about65% to about 97%, from about 70% to about 98%, or from about 80% toabout 99%.

In certain embodiments, the raw chitosan has two or more of thefollowing features, a Mw of from about 100 kDa to about 500 kDa, anumber-average molecular weight (Mn) in the range of from about 40 kDato about 300 kDa, a PDI of from about 1.7 to about 4.0 (Mw/Mn), a zaverage molecular weight (Mz) of from about 250 kDa to about 750 kDa, aPDI from about 1.5 to about 4.0 (Mz/Mw), and a degree of deacetylationof from about 65% to about 100%.

In certain embodiments, during step (b), the first chitosan intermediateis contacted with the acrylic acid at a temperature of about 50° C.,about 60° C., about 70° C., about 80° C., about 90° C., about 100° C.,about 110° C., or about 120° C. In certain embodiments, during step (b),the first chitosan intermediate is contacted with the acrylic acid at atemperature from about 50° C. to about 120° C., from about 60° C. toabout 120° C., from about 70° C. to about 120° C., from about 80° C. toabout 120° C., from about 90° C. to about 120° C., from about 100° C. toabout 120° C., from about 110° C. to about 120° C., from about 50° C. toabout 110° C., from about 50° C. to about 100° C., from about 50° C. toabout 90° C., from about 50° C. to about 80° C., from about 50° C. toabout 70° C., from about 50° C. to about 60° C., from about 60° C. toabout 110° C., from about 60° C. to about 100° C., from about 60° C. toabout 90° C., from about 60° C. to about 80° C., from about 60° C. toabout 70° C., from about 70° C. to about 110° C., from about 70° C. toabout 100° C., from about 70° C. to about 90° C., from about 70° C. toabout 80° C., from about 80° C. to about 110° C., from about 80° C. toabout 100° C., from about 80° C. to about 90° C., from about 90° C. toabout 110° C., from about 90° C. to about 100° C., or from about 100° C.to about 110° C. In certain embodiments, during step (b), the firstchitosan intermediate is contacted with the acrylic acid at atemperature from about 50° C. to about 100° C.

In certain embodiments, the acrylated chitosan intermediate formed instep (b) has a degree of substitution (DS) value of from about 25% toabout 65%.

In certain embodiments, during step (c), the acrylated chitosanintermediate is purified using a method selected from the groupconsisting of dialysis, gel filtration chromatography, stirred cellfiltration, tangential flow filtration, and any combination thereof.

In certain embodiments, the yield of acrylated chitosan obtainedfollowing step (c) is from about 80% to about 100%, from about 85% toabout 100%, from about 90% to about 100%, from about 95% to about 100%,from about 80% to about 95%, from about 80% to about 90%, from about 80%to about 85%, from about 85% to about 95%, from about 85% to about 90%,or from about 90% to about 95%. In certain embodiments, the yield ofacrylated chitosan obtained following step (c) is from about 80% toabout 97%.

In certain embodiments, the method further comprises the step ofprecipitating and/or lyophilizing the acrylated chitosan composition toprovide a solid.

In certain embodiments, the method further comprises the step ofdissolving the solid into an aqueous medium to form a solutioncomprising an amount of the acrylated chitosan composition of, forexample, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%,about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%,about 20%, about 21%, about 22%, about 23%, about 24%, or about 25% byweight of the total weight of the solution.

In certain embodiments, the amount of the acrylated chitosan compositioncomprises from about 1% to about 25%, from about 1% to about 20%, fromabout 1% to about 15%, from about 1% to about 10%, from about 1% toabout 9%, from about 1% to about 8%, from about 1% to about 7%, fromabout 1% to about 6%, from about 1% to about 5%, from about 1% to about4%, from about 1% to about 3%, from about 1% to about 2%, from about 2%to about 10%, from about 2% to about 9%, from about 2% to about 8%, fromabout 2% to about 7%, from about 2% to about 6%, from about 2% to about5%, from about 2% to about 4%, from about 2% to about 3%, from about 3%to about 10%, from about 3% to about 9%, from about 3% to about 8%, fromabout 3% to about 7%, from about 3% to about 6%, from about 3% to about5%, from about 3% to about 4%, from about 4% to about 10%, from about 4%to about 9%, from about 4% to about 8%, from about 4% to about 7%, fromabout 4% to about 6%, from about 4% to about 5%, from about 5% to about10%, from about 5% to about 9%, from about 5% to about 8%, from about 5%to about 7%, from about 5% to about 6%, from about 6% to about 10%, fromabout 6% to about 9%, from about 6% to about 8%, from about 6% to about7%, from about 7% to about 10%, from about 7% to about 9%, from about 7%to about 8%, from about 8% to about 10%, from about 8% to about 9%, orfrom about 9% to about 10%, by weight of the total weight of thesolution.

In certain embodiments, the amount of the acrylated chitosan compositioncomprises from about 1% to about 25% by weight of the total weight ofthe solution. In certain embodiments, the amount of the acrylatedchitosan composition comprises from about 1% to about 10% by weight ofthe total weight of the solution. In certain embodiments, the amount ofthe acrylated chitosan composition comprises from about 2% to about 5%,from about 4% to about 7%, or from about 5% to about 9%, by weight ofthe total weight of the solution.

In another aspect, provided herein is a method of preparing an acrylatedchitosan composition comprising:

-   -   (a) contacting a raw chitosan material with an acetic acid        solution to form a chitosan intermediate, wherein the raw        chitosan material has a PDI of from about 1.5 (Mw/Mn) to about        5.0 (Mw/Mn);    -   (b) contacting the intermediate chitosan with acrylic acid to        form an acrylated chitosan intermediate having a degree of        substitution (DS) value of from about 25% to about 65%; and    -   (c) purifying the acrylated chitosan intermediate to produce an        acrylated chitosan composition of the present invention.

In certain embodiments, the yield of acrylated chitosan obtainedfollowing step (c) is from about 80% to about 97%.

In certain embodiments, the method further comprising the step ofprecipitating the acrylated chitosan composition produced in step (c) toprovide a solid. In certain embodiments, the method further comprisingthe step of lyophilizing the acrylated chitosan composition to provide asolid.

In certain embodiments, the method further comprising the step ofdissolving the solid into an aqueous medium to form a solutioncomprising from about 1% to about 10% acrylated chitosan composition(w/w) of the total weight of the solution.

IV Oxidized Dextran Compositions

One aspect of the invention provides oxidized dextran compositions. Incertain embodiments, the oxidized dextran compositions comprise anoxidized dextran comprising:

-   -   (i) less than about 0.8 mole fraction of a first monomer of        formula (V)

and

-   -   (ii) about 0.1 to about 1.0 mole fraction of a second monomer,        wherein the second monomer is selected from a monomer of formula        (VI), a monomer of formula (VII) and a combination of        formula (VI) and formula (VII)

In certain embodiments, the oxidized dextran comprises from about 0.5 toabout 0.7, from about 0.52 to about 0.7, from about 0.54 to about 0.7,from about 0.56 to about 0.7, from about 0.58 to about 0.7, from about0.6 to about 0.7, from about 0.62 to about 0.7, from about 0.64 to about0.7, from about 0.66 to about 0.7, from about 0.68 to about 0.7, fromabout 0.5 to about 0.68, from about 0.5 to about 0.66, from about 0.5 toabout 0.64, from about 0.5 to about 0.62, from about 0.5 to about 0.6,from about 0.5 to about 0.58, from about 0.5 to about 0.56, from about0.5 to about 0.54, from about 0.5 to about 0.52, from about 0.52 toabout 0.68, from about 0.52 to about 0.66, from about 0.52 to about0.64, from about 0.52 to about 0.62, from about 0.52 to about 0.6, fromabout 0.52 to about 0.58, from about 0.52 to about 0.56, from about 0.52to about 0.54, from about 0.54 to about 0.68, from about 0.54 to about0.66, from about 0.54 to about 0.64, from about 0.54 to about 0.62, fromabout 0.54 to about 0.6, from about 0.54 to about 0.58, from about 0.54to about 0.56, from about 0.56 to about 0.68, from about 0.56 to about0.66, from about 0.56 to about 0.64, from about 0.56 to about 0.62, fromabout 0.56 to about 0.6, from about 0.56 to about 0.58, from about 0.58to about 0.68, from about 0.58 to about 0.66, from about 0.58 to about0.64, from about 0.58 to about 0.62, from about 0.58 to about 0.6, fromabout 0.6 to about 0.68, from about 0.6 to about 0.66, from about 0.6 toabout 0.64, from about 0.6 to about 0.62, from about 0.62 to about 0.68,from about 0.62 to about 0.66, from about 0.62 to about 0.64, from about0.64 to about 0.68, from about 0.64 to about 0.66, or from about 0.66 toabout 0.68 mole fraction of the first monomer of formula (V). In certainembodiments, the oxidized dextran comprises from about 0.4 to about 0.7mole fraction of the first monomer of formula (V). In certainembodiments, the oxidized dextran comprises from about 0.52 to about0.6, about 0.56 to about 0.62, or about 0.6 to about 0.66 mole fractionof the first monomer of formula (V).

In certain embodiments, the oxidized dextran comprises from about 0.15to about 0.35, from about 0.2 to about 0.35, from about 0.25 to about0.35, from about 0.3 to about 0.35, from about 0.15 to about 0.3, fromabout 0.15 to about 0.25, from about 0.15 to about 0.2, from about 0.2to about 0.3, from about 0.2 to about 0.25, or from about 0.25 to about0.3 mole fraction of the second monomer. In certain embodiments, theoxidized dextran comprises from about 0.15 to about 0.35 mole fractionof the second monomer. In certain embodiments, the oxidized dextrancomprises from about 0.15 to about 0.25, about 0.2 to about 0.3, orabout 0.25 to about 0.35 mole fraction of the second monomer.

In certain embodiments, wherein the oxidized dextran further comprisesless than about 0.65 mole fraction of a third monomer of formula (VIII)

In certain embodiments, the oxidized dextran comprises from about 0.1 toabout 0.16, from about 0.11 to about 0.16, from about 0.12 to about0.16, from about 0.13 to about 0.16, from about 0.14 to about 0.16, fromabout 0.15 to about 0.16, from about 0.1 to about 0.15, from about 0.1to about 0.14, from about 0.1 to about 0.13, from about 0.1 to about0.12, from about 0.1 to about 0.11, from about 0.11 to about 0.15, fromabout 0.11 to about 0.14, from about 0.11 to about 0.13, from about 0.11to about 0.12, from about 0.12 to about 0.15, from about 0.12 to about0.14, from about 0.12 to about 0.13, from about 0.13 to about 0.15, fromabout 0.13 to about 0.14, or from about 0.14 to about 0.15 mole fractionof a third monomer of formula (VIII). In certain embodiments, theoxidized dextran comprises from about 0.1 to about 0.16 mole fraction ofa third monomer of formula (VIII). In certain embodiments, the oxidizeddextran comprises from about 0.11 to about 0.14, about 0.12 to about0.15, or about 0.13 to about 0.15 mole fraction of a third monomer offormula (VIII).

In certain embodiments, wherein the oxidized dextran has a Mw of fromabout 10 kDa to about 300 kDa, about 10 kDa to about 200 kDa, about 15kDa to about 300 kDa, about 15 kDa to about 200 kDa, from about 15 kDato about 90 kDa, from about 15 kDa to about 80 kDa, from about 15 kDa toabout 70 kDa, from about 15 kDa to about 80 kDa, from about 15 kDa toabout 70 kDa, from about 15 kDa to about 60 kDa, from about 15 kDa toabout 50 kDa, from about 15 kDa to about 40 kDa, from about 15 kDa toabout 30 kDa, from about 15 kDa to about 25 kDa, from about 15 kDa toabout 20 kDa, from about 20 kDa to about 90 kDa, from about 20 kDa toabout 80 kDa, from about 20 kDa to about 70 kDa, from about 20 kDa toabout 60 kDa, from about 20 kDa to about 70 kDa, from about 20 kDa toabout 60 kDa, from about 20 kDa to about 50 kDa, from about 20 kDa toabout 40 kDa, from about 20 kDa to about 30 kDa, from about 20 kDa toabout 25 kDa, from about 25 kDa to about 90 kDa, from about 25 kDa toabout 80 kDa, from about 25 kDa to about 70 kDa, from about 25 kDa toabout 60 kDa, from about 25 kDa to about 70 kDa, from about 25 kDa toabout 60 kDa, from about 25 kDa to about 50 kDa, from about 25 kDa toabout 40 kDa, from about 25 kDa to about 30 kDa, from about 30 kDa toabout 90 kDa, from about 30 kDa to about 80 kDa, from about 30 kDa toabout 70 kDa, from about 30 kDa to about 60 kDa, from about 30 kDa toabout 50 kDa, from about 30 kDa to about 40 kDa, from about 40 kDa toabout 90 kDa, from about 40 kDa to about 80 kDa, from about 40 kDa toabout 70 kDa, from about 40 kDa to about 60 kDa, from about 40 kDa toabout 50 kDa, from about 50 kDa to about 90 kDa, from about 50 kDa toabout 80 kDa, from about 50 kDa to about 70 kDa, from about 50 kDa toabout 60 kDa, from about 60 kDa to about 90 kDa, from about 60 kDa toabout 80 kDa, from about 60 kDa to about 70 kDa, from about 70 kDa toabout 90 kDa, from about 70 kDa to about 80 kDa, or from about 80 kDa toabout 90 kDa.

In certain embodiments, wherein the oxidized dextran has a Mw of fromabout 10 kDa to about 300 kDa. In certain embodiments, wherein theoxidized dextran has a Mw of from about 15 kDa to about 90 kDa. Incertain embodiments, wherein the oxidized dextran has a Mw of from about15 kDa to about 20 kDa, from about 15 kDa to about 25 kDa or from about20 kDa to about 50 kDa.

In certain embodiments, the oxidized dextran has a Mn of from about 4kDa to about 166 kDa, about 4 kDa to about 100 kDa, from about 4 kDa toabout 50 kDa, from about 4 kDa to about 40 kDa, from about 4 kDa toabout 30 kDa, from about 4 kDa to about 20 kDa, from about 4 kDa toabout 10 kDa, from about 4 kDa to about 9 kDa, from about 4 kDa to about8 kDa, from about 4 kDa to about 7 kDa, from about 4 kDa to about 6 kDa,from about 4 kDa to about 5 kDa, from about 5 kDa to about 50 kDa, fromabout 5 kDa to about 40 kDa, from about 5 kDa to about 30 kDa, fromabout 5 kDa to about 20 kDa, from about 5 kDa to about 10 kDa, fromabout 5 kDa to about 9 kDa, from about 5 kDa to about 8 kDa, from about5 kDa to about 7 kDa, from about 5 kDa to about 6 kDa, from about 6 kDato about 50 kDa, from about 6 kDa to about 40 kDa, from about 6 kDa toabout 30 kDa, from about 6 kDa to about 20 kDa, from about 6 kDa toabout 10 kDa, from about 6 kDa to about 9 kDa, from about 6 kDa to about8 kDa, from about 6 kDa to about 7 kDa, from about 7 kDa to about 50kDa, from about 7 kDa to about 40 kDa, from about 7 kDa to about 30 kDa,from about 7 kDa to about 20 kDa, from about 7 kDa to about 10 kDa, fromabout 7 kDa to about 9 kDa, from about 7 kDa to about 8 kDa, from about8 kDa to about 50 kDa, from about 8 kDa to about 40 kDa, from about 8kDa to about 30 kDa, from about 8 kDa to about 20 kDa, from about 8 kDato about 10 kDa, from about 8 kDa to about 9 kDa, from about 9 kDa toabout 50 kDa, from about 9 kDa to about 40 kDa, from about 9 kDa toabout 30 kDa, from about 9 kDa to about 20 kDa, from about 9 kDa toabout 10 kDa, from about 10 kDa to about 50 kDa, from about 10 kDa toabout 40 kDa, from about 10 kDa to about 30 kDa, from about 10 kDa toabout 20 kDa, from about 20 kDa to about 50 kDa, from about 20 kDa toabout 40 kDa, from about 20 kDa to about 30 kDa, from about 30 kDa toabout 50 kDa, from about 30 kDa to about 40 kDa, or from about 40 kDa toabout 50 kDa.

In certain embodiments, the oxidized dextran has a Mn of from about 4kDa to about 166 kDa. In certain embodiments, the oxidized dextran has aMn of from about 4 kDa to about 45 kDa. In certain embodiments, theoxidized dextran has a Mn of from about 4 kDa to about 6 kDa, from about4 kDa to about 7 kDa, or from about 5 kDa to about 10 kDa.

In certain embodiments, the oxidized dextran has a polydispersity index(PDI) of from about 1.8 (Mw/Mn) to about 5.0 (Mw/Mn), from about 1.8(Mw/Mn) to about 6.0 (Mw/Mn), from about 2.0 (Mw/Mn) to about 6.0(Mw/Mn), from about 2.0 (Mw/Mn) to about 4.0 (Mw/Mn), from about 2.0(Mw/Mn) to about 3.8 (Mw/Mn), from about 2.0 (Mw/Mn) to about 3.6(Mw/Mn), from about 2.0 (Mw/Mn) to about 3.4 (Mw/Mn), from about 2.0(Mw/Mn) to about 3.2 (Mw/Mn), from about 2.0 (Mw/Mn) to about 3.0(Mw/Mn), from about 2.0 (Mw/Mn) to about 2.8 (Mw/Mn), from about 2.0(Mw/Mn) to about 2.6 (Mw/Mn), from about 2.0 (Mw/Mn) to about 2.4(Mw/Mn), from about 2.0 (Mw/Mn) to about 2.2 (Mw/Mn), from about 2.2(Mw/Mn) to about 4.0 (Mw/Mn), from about 2.2 (Mw/Mn) to about 3.8(Mw/Mn), from about 2.2 (Mw/Mn) to about 3.6 (Mw/Mn), from about 2.2(Mw/Mn) to about 3.4 (Mw/Mn), from about 2.2 (Mw/Mn) to about 3.2(Mw/Mn), from about 2.2 (Mw/Mn) to about 3.0 (Mw/Mn), from about 2.2(Mw/Mn) to about 2.8 (Mw/Mn), from about 2.2 (Mw/Mn) to about 2.6(Mw/Mn), from about 2.2 (Mw/Mn) to about 2.4 (Mw/Mn), from about 2.6(Mw/Mn) to about 4.0 (Mw/Mn), from about 2.6 (Mw/Mn) to about 3.8(Mw/Mn), from about 2.6 (Mw/Mn) to about 3.6 (Mw/Mn), from about 2.6(Mw/Mn) to about 3.4 (Mw/Mn), from about 2.6 (Mw/Mn) to about 3.2(Mw/Mn), from about 2.6 (Mw/Mn) to about 3.0 (Mw/Mn), from about 2.6(Mw/Mn) to about 2.8 (Mw/Mn), from about 2.8 (Mw/Mn) to about 4.0(Mw/Mn), from about 2.8 (Mw/Mn) to about 3.8 (Mw/Mn), from about 2.8(Mw/Mn) to about 3.6 (Mw/Mn), from about 2.8 (Mw/Mn) to about 3.4(Mw/Mn), from about 2.8 (Mw/Mn) to about 3.2 (Mw/Mn), from about 2.8(Mw/Mn) to about 3.0 (Mw/Mn), from about 3.0 (Mw/Mn) to about 4.0(Mw/Mn), from about 3.0 (Mw/Mn) to about 3.8 (Mw/Mn), from about 3.0(Mw/Mn) to about 3.6 (Mw/Mn), from about 3.0 (Mw/Mn) to about 3.4(Mw/Mn), from about 3.0 (Mw/Mn) to about 3.2 (Mw/Mn), from about 3.2(Mw/Mn) to about 4.0 (Mw/Mn), from about 3.2 (Mw/Mn) to about 3.8(Mw/Mn), from about 3.2 (Mw/Mn) to about 3.6 (Mw/Mn), from about 3.2(Mw/Mn) to about 3.4 (Mw/Mn), from about 3.4 (Mw/Mn) to about 4.0(Mw/Mn), from about 3.4 (Mw/Mn) to about 3.8 (Mw/Mn), from about 3.4(Mw/Mn) to about 3.6 (Mw/Mn), from about 3.6 (Mw/Mn) to about 4.0(Mw/Mn), from about 3.6 (Mw/Mn) to about 3.8 (Mw/Mn), or from about 3.8(Mw/Mn) to about 4.0 (Mw/Mn).

In certain embodiments, the oxidized dextran has a polydispersity index(PDI) of from about 1.8 (Mw/Mn) to about 6.0 (Mw/Mn). In certainembodiments, the oxidized dextran has a polydispersity index (PDI) offrom about 2.0 (Mw/Mn) to about 4.0 (Mw/Mn). In certain embodiments, theoxidized dextran has a polydispersity index (PDI) of from about 2.8(Mw/Mn) to about 3.4 (Mw/Mn), from about 3.2 (Mw/Mn) to about 3.6(Mw/Mn), or from about 3.4 (Mw/Mn) to about 4.0 (Mw/Mn).

In certain embodiments, the oxidized dextran has a Mz of from about 15kDa to about 500 kDa, from about 20 kDa to about 450 kDa, from about 20kDa to about 400 kDa, from about 20 kDa to about 300 kDa, from about 20kDa to about 200 kDa, from about 20 kDa to about 100 kDa, from about 20kDa to about 50 kDa, from about 20 kDa to about 45 kDa, from about 20kDa to about 40 kDa, from about 20 kDa to about 35 kDa, from about 20kDa to about 30 kDa, from about 20 kDa to about 25 kDa, from about 25kDa to about 450 kDa, from about 25 kDa to about 400 kDa, from about 25kDa to about 300 kDa, from about 25 kDa to about 200 kDa, from about 25kDa to about 100 kDa, from about 25 kDa to about 50 kDa, from about 25kDa to about 45 kDa, from about 25 kDa to about 40 kDa, from about 25kDa to about 35 kDa, from about 25 kDa to about 30 kDa, from about 30kDa to about 450 kDa, from about 30 kDa to about 400 kDa, from about 30kDa to about 300 kDa, from about 30 kDa to about 200 kDa, from about 30kDa to about 100 kDa, from about 30 kDa to about 50 kDa, from about 30kDa to about 45 kDa, from about 30 kDa to about 40 kDa, from about 30kDa to about 35 kDa, from about 35 kDa to about 450 kDa, from about 35kDa to about 400 kDa, from about 35 kDa to about 300 kDa, from about 35kDa to about 200 kDa, from about 35 kDa to about 100 kDa, from about 35kDa to about 50 kDa, from about 35 kDa to about 45 kDa, from about 35kDa to about 40 kDa, from about 40 kDa to about 450 kDa, from about 40kDa to about 400 kDa, from about 40 kDa to about 300 kDa, from about 40kDa to about 200 kDa, from about 40 kDa to about 100 kDa, from about 40kDa to about 50 kDa, from about 40 kDa to about 45 kDa, from about 45kDa to about 450 kDa, from about 45 kDa to about 400 kDa, from about 45kDa to about 300 kDa, from about 45 kDa to about 200 kDa, from about 45kDa to about 100 kDa, from about 45 kDa to about 50 kDa, from about 50kDa to about 450 kDa, from about 50 kDa to about 400 kDa, from about 50kDa to about 300 kDa, from about 50 kDa to about 200 kDa, from about 50kDa to about 100 kDa, from about 100 kDa to about 450 kDa, from about100 kDa to about 400 kDa, from about 100 kDa to about 300 kDa, fromabout 100 kDa to about 200 kDa, from about 200 kDa to about 450 kDa, 200kDa to about 400 kDa, from about 200 kDa to about 300 kDa, from about300 kDa to about 450 kDa, from about 300 kDa to about 400 kDa, or fromabout 400 kDa to about 450 kDa.

In certain embodiments, the oxidized dextran has a Mz of from about 15kDa to about 500 kDa. In certain embodiments, the oxidized dextran has aMz of from about 20 kDa to about 450 kDa. In certain embodiments, theoxidized dextran has a Mz of from about 20 kDa to about 35 kDa, about 30kDa to about 45 kDa or about 40 kDa to about 100 kDa.

In certain embodiments, the oxidized dextran has a polydispersity index(PDI) of from about 1.5 (Mz/Mw) to about 6.0 (Mz/Mw), from about 1.5(Mz/Mw) to about 5.0 (Mz/Mw), from about 1.5 (Mz/Mw) to about 4.0(Mz/Mw), from about 1.5 (Mz/Mw) to about 3.5 (Mz/Mw), from about 1.5(Mz/Mw) to about 3.0 (Mz/Mw), from about 1.5 (Mz/Mw) to about 2.5(Mz/Mw), from about 1.5 (Mz/Mw) to about 2.0 (Mz/Mw), from about 2.0(Mz/Mw) to about 5.0 (Mz/Mw), from about 2.0 (Mz/Mw) to about 4.0(Mz/Mw), from about 2.0 (Mz/Mw) to about 3.5 (Mz/Mw), from about 2.0(Mz/Mw) to about 3.0 (Mz/Mw), from about 2.0 (Mz/Mw) to about 2.5(Mz/Mw), from about 2.5 (Mz/Mw) to about 5.0 (Mz/Mw), from about 2.5(Mz/Mw) to about 4.0 (Mz/Mw), from about 2.5 (Mz/Mw) to about 3.5(Mz/Mw), from about 2.5 (Mz/Mw) to about 3.0 (Mz/Mw), from about 3.0(Mz/Mw) to about 5.0 (Mz/Mw), from about 3.0 (Mz/Mw) to about 4.0(Mz/Mw), from about 3.0 (Mz/Mw) to about 3.5 (Mz/Mw), from about 3.5(Mz/Mw) to about 5.0 (Mz/Mw), from about 3.5 (Mz/Mw) to about 4.0(Mz/Mw), or from about 4.0 (Mz/Mw) to about 5.0 (Mz/Mw).

In certain embodiments, the oxidized dextran has a polydispersity index(PDI) of from about 1.5 (Mz/Mw) to about 6.0 (Mz/Mw). In certainembodiments, the oxidized dextran has a polydispersity index (PDI) offrom about 1.5 (Mz/Mw) to about 5.0 (Mz/Mw). In certain embodiments, theoxidized dextran has a polydispersity index (PDI) of from about 1.5(Mz/Mw) to about 2.0 (Mz/Mw), about 1.5 (Mz/Mw) to about 2.5 (Mz/Mw) orabout 2.0 (Mz/Mw) to about 4.0 (Mz/Mw).

In certain embodiments, the oxidized dextran comprises a total amount ofaldehyde groups of from about 0.5 to about 2.0, from about 0.5 to about1.0, from about 0.6 to about 0.9, from about 0.6 to about 0.8, fromabout 0.6 to about 0.7, from about 0.7 to about 1.8, from about 0.7 toabout 1.6, from about 0.7 to about 1.4, from about 0.7 to about 1.2,from about 0.7 to about 1.0, from about 0.7 to about 0.9, from about 0.7to about 0.8, from about 0.8 to about 1.8, from about 0.8 to about 1.6,from about 0.8 to about 1.4, from about 0.8 to about 1.2, from about 0.8to about 1.0, from about 0.8 to about 0.9, from about 0.9 to about 1.8,from about 0.9 to about 1.6, from about 0.9 to about 1.4, from about 0.9to about 1.2, from about 0.9 to about 1.0, from about 1.0 to about 1.8,from about 1.0 to about 1.6, from about 1.0 to about 1.4, from about 1.0to about 1.2, from about 1.2 to about 1.8, from about 1.2 to about 1.6,from about 1.2 to about 1.4, from about 1.4 to about 1.8, from about 1.4to about 1.6, or from about 1.4 to about 1.8 mol/mol oxidized dextran.In certain embodiments, the oxidized dextran comprises a total amount ofaldehyde groups of from about 0.5 to about 2.0 mol/mol oxidized dextran.In certain embodiments, the oxidized dextran comprises a total amount ofaldehyde groups of from about 0.6 to about 0.9 mol/mol oxidized dextran.In certain embodiments, the oxidized dextran comprises a total amount ofaldehyde groups of from about 0.6 to about 0.8, from about 0.7 to about0.9, or from about 0.8 to about 1.4 mol/mol oxidized dextran.

In certain embodiments, the oxidized dextran comprises a total amount ofprimary aldehyde groups of from about 0.35 to about 2.0, about 0.35 toabout 1.0, about 0.35 to about 0.7, about 0.35 to about 0.6, about 0.35to about 0.55, about 0.35 to about 0.5, about 0.35 to about 0.45, about0.35 to about 0.4, about 0.4 to about 1.5, about 0.4 to about 1.0, about0.4 to about 0.6, about 0.4 to about 0.55, about 0.4 to about 0.5, about0.4 to about 0.45, about 0.45 to about 1.5, about 0.45 to about 1.0,about 0.45 to about 0.7, about 0.45 to about 0.6, about 0.45 to about0.55, about 0.45 to about 0.5, about 0.5 to about 1.5, about 0.5 toabout 1.0, about 0.5 to about 0.7, about 0.5 to about 0.6, about 0.5 toabout 0.55, about 0.55 to about 1.5, about 0.55 to about 1.0, about 0.55to about 0.7, about 0.55 to about 0.6, about 0.6 to about 1.5, about 0.6to about 1.0, about 0.6 to about 0.7, about 0.7 to about 1.5, about 0.7to about 1.0, or about 1.0 to about 1.5 mol/mol oxidized dextran. Incertain embodiments, the oxidized dextran comprises a total amount ofprimary aldehyde groups of from about 0.35 to about 2.0 mol/mol oxidizeddextran. In certain embodiments, the oxidized dextran comprises a totalamount of primary aldehyde groups of from about 0.35 to about 0.7mol/mol oxidized dextran. In certain embodiments, the oxidized dextrancomprises a total amount of primary aldehyde groups of from about 0.4 toabout 0.55, from about 0.45 to about 0.6, or from about 0.55 to about0.65 mol/mol oxidized dextran.

In certain embodiments, the oxidized dextran comprises a total amount ofsecondary aldehyde groups of from about 0.0 to about 1.3, from about 0.1to about 1.0, from about 0.1 to about 0.7, from about 0.1 to about 0.5,from about 0.1 to about 0.4, from about 0.1 to about 0.3, from about 0.1to about 0.2, from about 0.2 to about 1.0, from about 0.2 to about 0.7,from about 0.2 to about 0.5, from about 0.2 to about 0.4, from about 0.2to about 0.3, from about 0.3 to about 1.0, from about 0.3 to about 0.7,from about 0.3 to about 0.5, from about 0.3 to about 0.4, from about 0.4to about 1.0, from about 0.4 to about 0.7, from about 0.4 to about 0.5,from about 0.5 to about 1.0, from about 0.5 to about 0.7, or from about0.7 to about 1.0 mol/mol oxidized dextran. In certain embodiments, theoxidized dextran comprises a total amount of secondary aldehyde groupsof from about 0.0 to about 1.3 mol/mol oxidized dextran. In certainembodiments, the oxidized dextran comprises a total amount of secondaryaldehyde groups of from about 0.1 to about 0.3 mol/mol oxidized dextran.In certain embodiments, the oxidized dextran comprises a total amount ofsecondary aldehyde groups of from about 0.1 to about 0.3, about 0.2 toabout 0.3 or about 0.2 to about 0.5 mol/mol oxidized dextran.

In certain embodiments, the ratio of primary aldehyde groups tosecondary aldehyde groups is from about 1.8 to about 6.0, from about 1.8to about 4.0, from about 1.8 to about 3.5, from about 1.8 to about 3.25,from about 1.8 to about 3.0, from about 1.8 to about 2.75, from about1.8 to about 2.5, from about 1.8 to about 2.25, from about 1.8 to about2.0, from about 2.0 to about 3.5, from about 2.0 to about 3.25, fromabout 2.0 to about 3.0, from about 2.0 to about 2.75, from about 2.0 toabout 2.5, from about 2.0 to about 2.25, from about 2.25 to about 3.5,from about 2.25 to about 3.25, from about 2.25 to about 3.0, from about2.25 to about 2.75, from about 2.25 to about 2.5, from about 2.5 toabout 3.5, from about 2.5 to about 3.25, from about 2.5 to about 3.0,from about 2.5 to about 2.75, from about 2.75 to about 3.5, from about2.75 to about 3.25, from about 2.75 to about 3.0, from about 3.0 toabout 3.5, from about 3.0 to about 3.25, or from about 3.25 to about3.5. In certain embodiments, the ratio of primary aldehyde groups tosecondary aldehyde groups is from about 1.8 to about 6.0 or from about1.8 to about 3.5. In certain embodiments, the ratio of primary aldehydegroups to secondary aldehyde groups is from about 1.8 to about 2.0,about 1.8 to about 2.25, or about 2.0 to about 3.25.

In certain embodiments, the degree of oxidation is from about 25% toabout 100%, from about 25% to about 90%, from about 25% to about 80%,from about 25% to about 70%, from about 25% to about 60%, from about 30%to about 45%, from about 30% to about 40%, from about 30% to about 35%,from about 35% to about 45%, from about 35% to about 40%. In certainembodiments, the degree of oxidation is from about 25% to about 100% orfrom about 30% to about 50%. In certain embodiments, the degree ofoxidation is from about 30% to about 45%, or about 35% to about 45%.

In certain embodiments, the oxidized dextran composition comprises about1% (w/w), about 2% (w/w), about 3% (w/w), about 4% (w/w), about 5%(w/w), about 6% (w/w), about 7% (w/w), about 8% (w/w), about 9% (w/w),about 10% (w/w), about 11% (w/w), about 12% (w/w), about 13% (w/w),about 14% (w/w), about 15% (w/w), about 16% (w/w), about 17% (w/w),about 18% (w/w), about 19% (w/w), about 20% (w/w), about 21% (w/w),about 22% (w/w), about 23% (w/w), about 24% (w/w), or about 25% (w/w) byweight of the total weight of the solution.

In certain embodiments, the oxidized dextran composition comprises fromabout 1% (w/w) to about 25% (w/w), from about 1% (w/w) to about 20%(w/w), from about 1% (w/w) to about 15% (w/w), from about 1% (w/w) toabout 10% (w/w), from about 1% (w/w) to about 9% (w/w), from about 1%(w/w) to about 8% (w/w), from about 1% (w/w) to about 7% (w/w), fromabout 1% (w/w) to about 6% (w/w), from about 1% (w/w) to about 5% (w/w),from about 1% (w/w) to about 4% (w/w), from about 1% (w/w) to about 3%(w/w), from about 1% (w/w) to about 2% (w/w), from about 2% (w/w) toabout 10% (w/w), from about 2% (w/w) to about 9% (w/w), from about 2%(w/w) to about 8% (w/w), from about 2% (w/w) to about 7% (w/w), fromabout 2% (w/w) to about 6% (w/w), from about 2% (w/w) to about 5% (w/w),from about 2% (w/w) to about 4% (w/w), from about 2% (w/w) to about 3%(w/w), from about 3% (w/w) to about 10% (w/w), from about 3% (w/w) toabout 9% (w/w), from about 3% (w/w) to about 8% (w/w), from about 3%(w/w) to about 7% (w/w), from about 3% (w/w) to about 6% (w/w), fromabout 3% (w/w) to about 5% (w/w), from about 3% (w/w) to about 4% (w/w),from about 4% (w/w) to about 10% (w/w), from about 4% (w/w) to about 9%(w/w), from about 4% (w/w) to about 8% (w/w), from about 4% (w/w) toabout 7% (w/w), from about 4% (w/w) to about 6% (w/w), from about 4%(w/w) to about 5% (w/w), from about 5% (w/w) to about 10% (w/w), fromabout 5% (w/w) to about 9% (w/w), from about 5% (w/w) to about 8% (w/w),from about 5% (w/w) to about 7% (w/w), from about 5% (w/w) to about 6%(w/w), from about 6% (w/w) to about 10% (w/w), from about 6% (w/w) toabout 9% (w/w), from about 6% (w/w) to about 8% (w/w), from about 6%(w/w) to about 7% (w/w), from about 7% (w/w) to about 10% (w/w), fromabout 7% (w/w) to about 9% (w/w), from about 7% (w/w) to about 8% (w/w),from about 8% (w/w) to about 10% (w/w), from about 8% (w/w) to about 9%(w/w), or from about 9% (w/w) to about 10% (w/w) of the oxidizeddextran. In certain embodiments, the oxidized dextran compositioncomprises from about 1% (w/w) to about 25% (w/w) or from about 1% (w/w)to about 10% (w/w) of the oxidized dextran. In certain embodiments, theoxidized dextran composition comprises about 2% (w/w) to about 5% (w/w),about 4% (w/w) to about 7% (w/w), or about 6% (w/w) to about 10% (w/w)of the oxidized dextran.

In certain embodiments, the oxidized dextran composition has a viscosityof from about 1.0 cP to about 2,000 cP, from about 1.0 cP to about 1,000cP, from about 1.0 cP to about 100 cP, from about 1.0 cP to about 10 cP,from about 1.0 cP to about 9 cP, from about 1.0 cP to about 8 cP, fromabout 1.0 cP to about 7 cP, from about 1.0 cP to about 6 cP, from about1.0 cP to about 5 cP, from about 1.0 cP to about 4 cP, from about 1.0 cPto about 3 cP, from about 1.0 cP to about 2 cP, from about 2.0 cP toabout 10.0 cP, from about 2.0 cP to about 9.0 cP, from about 2.0 cP toabout 8.0 cP, from about 2.0 cP to about 7.0 cP, from about 2.0 cP toabout 6.0 cP, from about 2.0 cP to about 5.0 cP, from about 2.0 cP toabout 4.0 cP, from about 2.0 cP to about 3.0 cP, from about 3.0 cP toabout 10.0 cP, from about 3.0 cP to about 9.0 cP, from about 3.0 cP toabout 8.0 cP, from about 3.0 cP to about 7.0 cP, from about 3.0 cP toabout 6.0 cP, from about 3.0 cP to about 5.0 cP, from about 3.0 cP toabout 4.0 cP, from about 4.0 cP to about 10.0 cP, from about 4.0 cP toabout 9.0 cP, from about 4.0 cP to about 8.0 cP, from about 4.0 cP toabout 7.0 cP, from about 4.0 cP to about 6.0 cP, from about 4.0 cP toabout 5.0 cP, from about 5.0 cP to about 10.0 cP, from about 5.0 cP toabout 9.0 cP, from about 5.0 cP to about 8.0 cP, from about 5.0 cP toabout 7.0 cP, from about 5.0 cP to about 6.0 cP, from about 6.0 cP toabout 10.0 cP, from about 6.0 cP to about 9.0 cP, from about 6.0 cP toabout 8.0 cP, from about 6.0 cP to about 7.0 cP, from about 7.0 cP toabout 10.0 cP, from about 7.0 cP to about 9.0 cP, from about 7.0 cP toabout 8.0 cP, from about 8.0 cP to about 10.0 cP, from about 8.0 cP toabout 9.0 cP, or from about 9.0 cP to about 10.0 cP.

In certain embodiments, the oxidized dextran composition has a viscosityof from about 1 cP to about 2,000 cP, or from about 1 cP to about 10 cP.In certain embodiments, the oxidized dextran composition has a viscosityof from about 1.0 cP to about 3.0 cP, about 1.0 cP to about 5.0 cP, orabout 3.0 cP to about 9.0 cP.

In certain embodiments, the oxidized dextran composition has a pH offrom about 5.0 to about 7.5, from about 5.2 to about 7.5, from about 5.4to about 7.5, from about 5.6 to about 7.5, from about 5.8 to about 7.5,from about 6.0 to about 7.5, from about 6.2 to about 7.5, from about 6.4to about 7.5, from about 6.6 to about 7.5, from about 6.8 to about 7.5,from about 7.0 to about 7.5, from about 5.0 to about 7.0, from about 5.0to about 6.8, from about 5.0 to about 6.6, from about 5.0 to about 6.4,from about 5.0 to about 6.2, from about 5.0 to about 6.0, from about 5.0to about 5.8, from about 5.0 to about 5.6, from about 5.0 to about 5.4,from about 5.0 to about 5.2, from about 5.2 to about 7.0, from about 5.2to about 6.8, from about 5.2 to about 6.6, from about 5.2 to about 6.4,from about 5.2 to about 6.2, from about 5.2 to about 6.0, from about 5.2to about 5.8, from about 5.2 to about 5.6, from about 5.2 to about 5.4,from about 5.4 to about 7.0, from about 5.4 to about 6.8, from about 5.4to about 6.6, from about 5.4 to about 6.4, from about 5.4 to about 6.2,from about 5.4 to about 6.0, from about 5.4 to about 5.8, from about 5.4to about 5.6, from about 5.6 to about 7.0, from about 5.6 to about 6.8,from about 5.6 to about 6.6, from about 5.6 to about 6.4, from about 5.6to about 6.2, from about 5.6 to about 6.0, from about 5.6 to about 5.8,from about 5.8 to about 7.0, from about 5.8 to about 6.8, from about 5.8to about 6.6, from about 5.8 to about 6.4, from about 5.8 to about 6.2,from about 5.8 to about 6.0, from about 6.0 to about 7.0, from about 6.0to about 6.8, from about 6.0 to about 6.6, from about 6.0 to about 6.4,from about 6.0 to about 6.2, from about 6.2 to about 7.0, from about 6.2to about 6.8, from about 6.2 to about 6.6, from about 6.2 to about 6.4,from about 6.4 to about 7.0, from about 6.4 to about 6.8, from about 6.4to about 6.6, from about 6.6 to about 7.0, from about 6.6 to about 6.8,or from about 6.8 to about 7.0. In certain embodiments, the oxidizeddextran composition has a pH of from about 5.0 to about 7.5. In certainembodiments, the oxidized dextran composition has a pH of from about 5.0to about 6.0, from about 5.0 to about 6.8, or from about 5.6 to about7.0.

In certain embodiments, the oxidized dextran composition has aconductivity of from about 0.05 (mS/cm) to about 1.7 (mS/cm), from about0.05 (mS/cm) to about 1.2 (mS/cm), from about 0.05 (mS/cm) to about 0.7(mS/cm), from about 0.05 (mS/cm) to about 0.5 (mS/cm), from about 0.05(mS/cm) to about 0.3 (mS/cm), from about 0.05 (mS/cm) to about 0.25(mS/cm), from about 0.05 (mS/cm) to about 0.2 (mS/cm), from about 0.05(mS/cm) to about 0.15 (mS/cm), from about 0.05 (mS/cm) to about 0.1(mS/cm), from about 0.1 (mS/cm) to about 0.7 (mS/cm), from about 0.1(mS/cm) to about 0.5 (mS/cm), from about 0.1 (mS/cm) to about 0.3(mS/cm), from about 0.1 (mS/cm) to about 0.25 (mS/cm), from about 0.1(mS/cm) to about 0.2 (mS/cm), from about 0.1 (mS/cm) to about 0.15(mS/cm), from about 0.15 (mS/cm) to about 0.7 (mS/cm), from about 0.15(mS/cm) to about 0.5 (mS/cm), from about 0.15 (mS/cm) to about 0.3(mS/cm), from about 0.15 (mS/cm) to about 0.25 (mS/cm), from about 0.15(mS/cm) to about 0.2 (mS/cm), from about 0.2 (mS/cm) to about 0.7(mS/cm), from about 0.2 (mS/cm) to about 0.5 (mS/cm), from about 0.2(mS/cm) to about 0.3 (mS/cm), from about 0.2 (mS/cm) to about 0.25(mS/cm), from about 0.25 (mS/cm) to about 0.7 (mS/cm), from about 0.25(mS/cm) to about 0.5 (mS/cm), from about 0.25 (mS/cm) to about 0.3(mS/cm), from about 0.3 (mS/cm) to about 0.7 (mS/cm), from about 0.3(mS/cm) to about 0.5 (mS/cm), or from about 0.5 (mS/cm) to about 0.7(mS/cm).

In certain embodiments, the oxidized dextran composition has aconductivity of from about 0.05 (mS/cm) to about 1.7 (mS/cm). In certainembodiments, the oxidized dextran composition has a conductivity of fromabout 0.05 (mS/cm) to about 0.7 (mS/cm). In certain embodiments, theoxidized dextran composition has a conductivity of from about from about0.1 (mS/cm) to about 0.3 (mS/cm), from about 0.2 (mS/cm) to about 0.5(mS/cm) or from about 0.3 (mS/cm) to about 0.7 (mS/cm).

In certain embodiments, the oxidized dextran has two or more propertiesselected from a Mw of from about 10 kDa to about 300 kDa, a Mn of fromabout 4 kDa to about 166 kDa, a PDI of from about 1.8 to about 6.0(Mw/Mn), a total amount of aldehyde groups of from about 0.5 to about2.0 mol aldehydes/mol oxidized dextran, a total amount of primaryaldehydes of from about 0.35 to about 2.0 mol aldehydes/mol oxidizeddextran, a total amount of secondary aldehydes of from about 0.0 toabout 1.3 mol aldehydes/mol oxidized dextran, a ratio of primaryaldehyde groups to secondary aldehyde groups from about 1.6 to about6.0, a degree of oxidation from about 25% to about 100%.

In certain embodiments, the oxidized dextran composition comprises aplurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) or all of thefollowing features: (i) a Mw of from about 15 kDa to about 90 kDa, (ii)a Mn of from about 4 kDa to about 45 kDa, (iii) a Mz of from about 22kDa to about 450 kDa, (iv) a PDI (Mw/Mn) of from about 2.0 to about 4.0,(v) a PDI (Mz/Mw) of from about 1.5 to about 5.0, (vi) a mole fractionof the monomer of formula (V) of from about 0.5 to about 0.7, (vii) amole fraction of the monomer of formula (VI) and/or the monomer offormula (VII) of from about 0.15 to about 0.35, (viii) a mole fractionof the monomer of formula (VIII) of from about 0.1 to about 0.16, (ix) atotal amount of aldehyde groups of from about 0.65 to about 0.9 molaldehydes/mol oxidized dextran, (x) a total amount of primary aldehydegroups of from about 0.38 to about 0.7 mol aldehydes/mol oxidizeddextran, (xi) a total amount of secondary aldehyde groups of from about0.1 to about 0.31 mol aldehydes/mol oxidized dextran, (xii) a ratio ofprimary aldehyde groups to secondary aldehyde groups of from about 1.8to about 3.5, and (xiii) a degree of oxidation of from about 32% toabout 45%.

In certain embodiments, the oxidized dextran composition comprises aplurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16)or all of the following features: (i) a Mw of from about 15 kDa to about90 kDa, (ii) a Mn of from about 4 kDa to about 45 kDa, (iii) a Mz offrom about 22 kDa to about 450 kDa, (iv) a PDI (Mw/Mn) of from about 2.0to about 4.0, (v) a PDI (Mz/Mw) of from about 1.5 to about 5.0, (vi) amole fraction of the monomer of formula (V) of from about 0.5 to about0.7, (vii) a mole fraction of the monomer of formula (VI) and/or themonomer of formula (VII) of from about 0.15 to about 0.35, (viii) a molefraction of the monomer of formula (VIII) of from about 0.1 to about0.16, (ix) a total amount of aldehyde groups of from about 0.65 to about0.9 mol aldehydes/mol oxidized dextran, (x) a total amount of primaryaldehyde groups of from about 0.38 to about 0.7 mol aldehydes/moloxidized dextran, (xi) a total amount of secondary aldehyde groups offrom about 0.1 to about 0.31 mol aldehydes/mol oxidized dextran, (xii) aratio of primary aldehyde groups to secondary aldehyde groups of fromabout 1.8 to about 3.5, (xiii) a degree of oxidation of from about 32%to about 45%, (xiv) a concentration of the acrylated chitosan in anaqueous medium or from about 1% (w/w) to about 10% (w/w), (xv) aviscosity of from about 1 cP to about 10 cP, (xvi) a pH of from about5.0 to about 7.5, and (xvii) a conductivity of from about 0.05 mS/cm toabout 0.7 mS/cm.

V Methods of Making Oxidized Dextran Compositions

Dextran is an α-D-1,6-glucose-linked glucan with side chains 1-3 linkedto the backbone units of the dextran biopolymer.

One aspect of the invention provides methods of preparing oxidizeddextran compositions. In various embodiments, the methods generallyinclude:

-   -   (a) contacting a raw dextran material with an oxidizing agent to        form an oxidized dextran intermediate; and    -   (b) purifying the oxidized dextran intermediate to produce an        oxidized dextran composition of the present invention.

In certain embodiments, during step (a), the raw dextran material has aMw of from about 50 kDa to about 2,000 kDa, from about 50 kDa to about1,000 kDa, from about 50 kDa to about 500 kDa, from about 50 kDa toabout 400 kDa, from about 60 kDa to about 2,000 kDa, from about 60 kDato about 1,000 kDa, from about 50 kDa to about 500 kDa, from about 60kDa to about 400 kDa, from about 60 kDa to about 300 kDa, from about 60kDa to about 200 kDa, from about 60 kDa to about 100 kDa, from about 60kDa to about 90 kDa, from about 60 kDa to about 80 kDa, from about 60kDa to about 70 kDa, from about 70 kDa to about 400 kDa, from about 70kDa to about 300 kDa, from about 70 kDa to about 200 kDa, from about 70kDa to about 100 kDa, from about 70 kDa to about 90 kDa, from about 70kDa to about 80 kDa, from about 80 kDa to about 400 kDa, from about 80kDa to about 300 kDa, from about 80 kDa to about 200 kDa, from about 80kDa to about 100 kDa, from about 80 kDa to about 90 kDa, from about 90kDa to about 400 kDa, from about 90 kDa to about 300 kDa, from about 90kDa to about 200 kDa, from about 90 kDa to about 100 kDa, from about 100kDa to about 400 kDa, from about 100 kDa to about 300 kDa, from about100 kDa to about 200 kDa, from about 200 kDa to about 400 kDa, fromabout 200 kDa to about 300 kDa, or from about 300 kDa to about 400 kDa.In certain embodiments, during step (a), the raw dextran material has aMw of from about 50 kDa to about 2,000 kDa. In certain embodiments,during step (a), the raw dextran material has a Mw of from about 60 kDato about 400 kDa. In certain embodiments, during step (a), the rawdextran material has a Mw of from about 60 kDa to about 100 kDa, about80 kDa to about 200 kDa, or about 100 kDa to about 400 kDa.

In certain embodiments, during step (a), the raw dextran material has aMn of from about 15 kDa to about 500 kDa, from about 15 kDa to about 250kDa, from about 25 kDa to about 500 kDa, from about 25 kDa to about 250kDa, from about 25 kDa to about 200 kDa, from about 25 kDa to about 160kDa, from about 25 kDa to about 140 kDa, from about 25 kDa to about 120kDa, from about 25 kDa to about 100 kDa, from about 25 kDa to about 90kDa, from about 25 kDa to about 80 kDa, from about 25 kDa to about 70kDa, from about 25 kDa to about 60 kDa, from about 25 kDa to about 50kDa, from about 25 kDa to about 40 kDa, from about 25 kDa to about 35kDa, from about 25 kDa to about 30 kDa, from about 30 kDa to about 250kDa, from about 30 kDa to about 200 kDa, from about 30 kDa to about 160kDa, from about 30 kDa to about 140 kDa, from about 30 kDa to about 120kDa, from about 30 kDa to about 100 kDa, from about 30 kDa to about 90kDa, from about 30 kDa to about 80 kDa, from about 30 kDa to about 70kDa, from about 30 kDa to about 60 kDa, from about 30 kDa to about 50kDa, from about 30 kDa to about 40 kDa, from about 30 kDa to about 35kDa, from about 35 kDa to about 250 kDa, from about 35 kDa to about 200kDa, from about 35 kDa to about 160 kDa, from about 35 kDa to about 140kDa, from about 35 kDa to about 120 kDa, from about 35 kDa to about 100kDa, from about 35 kDa to about 90 kDa, from about 35 kDa to about 80kDa, from about 35 kDa to about 70 kDa, from about 35 kDa to about 60kDa, from about 35 kDa to about 50 kDa, from about 35 kDa to about 40kDa, from about 40 kDa to about 250 kDa, from about 40 kDa to about 200kDa, from about 40 kDa to about 160 kDa, from about 40 kDa to about 140kDa, from about 40 kDa to about 120 kDa, from about 40 kDa to about 100kDa, from about 40 kDa to about 90 kDa, from about 40 kDa to about 80kDa, from about 40 kDa to about 70 kDa, from about 40 kDa to about 60kDa, from about 40 kDa to about 50 kDa, from about 50 kDa to about 250kDa, from about 50 kDa to about 200 kDa, from about 50 kDa to about 160kDa, from about 50 kDa to about 150 kDa, from about 50 kDa to about 120kDa, from about 50 kDa to about 100 kDa, from about 50 kDa to about 90kDa, from about 50 kDa to about 80 kDa, from about 50 kDa to about 70kDa, or from about 50 kDa to about 60 kDa.

In certain embodiments, during step (a), the raw dextran material has aMn of from about 15 kDa to about 500 kDa. In certain embodiments, duringstep (a), the raw dextran material has a Mn of from about 25 kDa toabout 250 kDa. In certain embodiments, during step (a), the raw dextranmaterial has a Mn of from about 25 kDa to about 120 kDa, from about 40kDa to about 140 kDa, or from about 80 kDa to about 200 kDa.

In certain embodiments, during step (a), the raw dextran material has aPDI of from about 1.3 (Mw/Mn) to about 5.0 (Mw/Mn), from about 1.4(Mw/Mn) to about 5.0 (Mw/Mn), from about 1.4 (Mw/Mn) to about 4.0(Mw/Mn), from about 1.4 (Mw/Mn) to about 3.0 (Mw/Mn), from about 1.5(Mw/Mn) to about 5.0 (Mw/Mn), from about 1.5 (Mw/Mn) to about 4.0(Mw/Mn), from about 1.5 (Mw/Mn) to about 3.0 (Mw/Mn), from about 1.5(Mw/Mn) to about 2.5 (Mw/Mn), from about 1.5 (Mw/Mn) to about 2.0(Mw/Mn), from about 1.5 (Mw/Mn) to about 1.9 (Mw/Mn), from about 1.5(Mw/Mn) to about 1.7 (Mw/Mn), from about 1.7 (Mw/Mn) to about 3.0(Mw/Mn), from about 1.7 (Mw/Mn) to about 2.5 (Mw/Mn), from about 1.7(Mw/Mn) to about 2.0 (Mw/Mn), from about 1.7 (Mw/Mn) to about 1.9(Mw/Mn), from about 1.9 (Mw/Mn) to about 3.0 (Mw/Mn), from about 1.9(Mw/Mn) to about 2.5 (Mw/Mn), or from about 1.9 (Mw/Mn) to about 2.0(Mw/Mn).

In certain embodiments, during step (a), the raw dextran material has aPDI of from about 1.4 (Mw/Mn) to about 5.0 (Mw/Mn). In certainembodiments, during step (a), the raw dextran material has a PDI of fromabout 1.5 (Mw/Mn) to about 3.0 (Mw/Mn). In certain embodiments, duringstep (a), the raw dextran material has a PDI of from about 1.5 (Mw/Mn)to about 2.0 (Mw/Mn), about 1.5 (Mw/Mn) to about 2.5 (Mw/Mn), or about1.9 (Mw/Mn) to about 3.0 (Mw/Mn).

In certain embodiments, during step (a), the raw dextran material has aMz of from about 250 kDa to about 5,000 kDa, from about 250 kDa to about4,000 kDa, from about 250 kDa to about 3,000 kDa, from about 250 kDa toabout 2,000 kDa, from about 300 kDa to about 5,000 kDa, from about 300kDa to about 4,000 kDa, from about 300 kDa to about 3,000 kDa, about 300kDa to about 2,000 kDa, from about 300 kDa to about 1,500 kDa, fromabout 300 kDa to about 1,000 kDa, from about 300 kDa to about 750 kDa,from about 300 kDa to about 500 kDa, from about 300 kDa to about 450kDa, from about 300 kDa to about 400 kDa, from about 300 kDa to about350 kDa, from about 350 kDa to about 2,000 kDa, from about 350 kDa toabout 1,500 kDa, from about 350 kDa to about 1,000 kDa, from about 350kDa to about 750 kDa, from about 350 kDa to about 500 kDa, from about350 kDa to about 450 kDa, from about 350 kDa to about 400 kDa, fromabout 400 kDa to about 2,000 kDa, from about 400 kDa to about 1,500 kDa,from about 400 kDa to about 1,000 kDa, from about 400 kDa to about 750kDa, from about 400 kDa to about 500 kDa, from about 400 kDa to about450 kDa, from about 450 kDa to about 2,000 kDa, from about 450 kDa toabout 1,500 kDa, from about 450 kDa to about 1,000 kDa, from about 450kDa to about 750 kDa, from about 450 kDa to about 500 kDa, from about500 kDa to about 2,000 kDa, from about 500 kDa to about 1,500 kDa, fromabout 500 kDa to about 1,000 kDa, from about 500 kDa to about 750 kDa,from about 750 kDa to about 2,000 kDa, from about 750 kDa to about 1,500kDa, from about 750 kDa to about 1,000 kDa, from about 1,000 kDa toabout 2,000 kDa, from about 1,000 kDa to about 1,500 kDa, or from about1,500 kDa to about 2,000 kDa.

In certain embodiments, during step (a), the raw dextran material has aMz of from about 250 kDa to about 5,000 kDa. In certain embodiments,during step (a), the raw dextran material has a Mz of from about 300 kDato about 2,000 kDa. In certain embodiments, during step (a), the rawdextran material has a Mz of from about 300 kDa to about 1,000 kDa, fromabout 350 kDa to about 1,500 kDa, or from about 500 kDa to about 2,000kDa.

In certain embodiments, during step (a), the raw dextran material has aPDI of from about 1.4 (Mz/Mw) to about 15.0 (Mz/Mw), from about 1.5(Mz/Mw) to about 15.0 (Mz/Mw), from about 1.7 (Mz/Mw) to about 15.0(Mz/Mw), from about 2.0 (Mz/Mw) to about 15.0 (Mz/Mw), from about 3.0(Mz/Mw) to about 15.0 (Mz/Mw), from about 6.0 (Mz/Mw) to about 15.0(Mz/Mw), from about 9.0 (Mz/Mw) to about 15.0 (Mz/Mw), from about 12.0(Mz/Mw) to about 15.0 (Mz/Mw), from about 1.5 (Mz/Mw) to about 12.0(Mz/Mw), from about 1.5 (Mz/Mw) to about 9.0 (Mz/Mw), from about 1.5(Mz/Mw) to about 6.0 (Mz/Mw), from about 1.5 (Mz/Mw) to about 3.0(Mz/Mw), from about 1.5 (Mz/Mw) to about 2.0 (Mz/Mw), from about 1.5(Mz/Mw) to about 1.7 (Mz/Mw), from about 1.7 (Mz/Mw) to about 12.0(Mz/Mw), from about 1.7 (Mz/Mw) to about 9.0 (Mz/Mw), from about 1.7(Mz/Mw) to about 6.0 (Mz/Mw), from about 1.7 (Mz/Mw) to about 3.0(Mz/Mw), from about 1.7 (Mz/Mw) to about 2.0 (Mz/Mw), from about 2.0(Mz/Mw) to about 12.0 (Mz/Mw), from about 2.0 (Mz/Mw) to about 9.0(Mz/Mw), from about 2.0 (Mz/Mw) to about 6.0 (Mz/Mw), from about 2.0(Mz/Mw) to about 3.0 (Mz/Mw), from about 3.0 (Mz/Mw) to about 12.0(Mz/Mw), from about 3.0 (Mz/Mw) to about 9.0 (Mz/Mw), from about 3.0(Mz/Mw) to about 6.0 (Mz/Mw), from about 6.0 (Mz/Mw) to about 12.0(Mz/Mw), from about 6.0 (Mz/Mw) to about 9.0 (Mz/Mw), or from about 9.0(Mz/Mw) to about 12.0 (Mz/Mw).

In certain embodiments, during step (a), the raw dextran material has aPDI of from about 1.4 (Mz/Mw) to about 15.0 (Mz/Mw). In certainembodiments, during step (a), the raw dextran material has a PDI of fromabout 1.5 (Mz/Mw) to about 15.0 (Mz/Mw). In certain embodiments, duringstep (a), the raw dextran material has a PDI of from about 1.5 (Mz/Mw)to about 9.0 (Mz/Mw), about 1.5 (Mz/Mw) to about 12.0 (Mz/Mw), or about6.0 (Mz/Mw) to about 15.0 (Mz/Mw).

In certain embodiments, the raw dextran material comprises two or moreof the features selected from an Mw of from about 50 kDa to about 2,000kDa, an Mn of from about 15 kDa to about 500 kDa, a PDI of from about1.4 to about 5.0 (Mw/Mn), a Mz of from about 250 kDa to about 5,000 kDa,and a PDI pf from about 1.4 to about 15.0 (Mz/Mw).

In certain embodiments, during step (a), the raw dextran material iscontacted with the oxidizing agent at a temperature of about 0° C.,about 5° C., about 10° C., about 15° C., about 20° C., about 25° C.,about 30° C., about 35° C., about 40° C., about 45° C., or about 50° C.In certain embodiments, in step (a), the raw dextran material iscontacted with the oxidizing agent at a temperature from about 0° C. toabout 50° C., from about 10° C. to about 50° C., from about 20° C. toabout 50° C., from about 30° C. to about 50° C., from about 40° C. toabout 50° C., from about 10° C. to about 40° C., from about 10° C. toabout 30° C., from about 10° C. to about 20° C., from about 20° C. toabout 40° C., from about 20° C. to about 30° C., or from about 30° C. toabout 40° C. In certain embodiments, in step (a), the raw dextranmaterial is contacted with the oxidizing agent at a temperature fromabout 0° C. to about 50° C.

In certain embodiments, during step (a), the oxidizing agent is aperiodate salt. In certain embodiments, the periodate salt is selectedfrom the group consisting of sodium periodate or potassium periodate.

In certain embodiments, during step (b), the oxidized dextran ispurified using a method selected from the group consisting of dialysis,gel filtration chromatography, stirred cell filtration, tangential flowfiltration, and combinations thereof.

In certain embodiments, the yield of oxidized dextran obtained followingstep (c) is from about 75% to about 100%, from about 80% to about 100%,from about 85% to about 100%, from about 90% to about 100%, from about95% to about 100%, from about 75% to about 95%, from about 75% to about90%, from about 75% to about 85%, from about 75% to about 80%, fromabout 80% to about 95%, from about 80% to about 90%, from about 80% toabout 85%, from about 85% to about 95%, from about 85% to about 90%, orfrom about 90% to about 95%. In certain embodiments, the yield ofacrylated chitosan obtained following step (c) is from about 75% toabout 95%.

In certain embodiments, the method further comprising the step ofprecipitating the oxidized dextran composition to provide a solid. Incertain embodiments, the method further comprising the step oflyophilizing the oxidized dextran composition to provide a solid.

In certain embodiments, the method further comprising the step ofdissolving the solid into an aqueous medium to form a solutioncomprising an amount of the oxidized dextran composition.

In certain embodiments, the amount of the oxidized dextran compositioncomprises about 1%, about 2%, about 3%, about 4%, about 5%, about 6%,about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%,about 20%, about 21%, about 22%, about 23%, about 24%, or about 25% byweight of the total weight of the solution.

In certain embodiments, the amount of the oxidized dextran compositioncomprises from about 1% to about 25%, from about 1% to about 20%, fromabout 1% to about 15%, from about 1% to about 10%, from about 1% toabout 9%, from about 1% to about 8%, from about 1% to about 7%, fromabout 1% to about 6%, from about 1% to about 5%, from about 1% to about4%, from about 1% to about 3%, from about 1% to about 2%, from about 2%to about 10%, from about 2% to about 9%, from about 2% to about 8%, fromabout 2% to about 7%, from about 2% to about 6%, from about 2% to about5%, from about 2% to about 4%, from about 2% to about 3%, from about 3%to about 10%, from about 3% to about 9%, from about 3% to about 8%, fromabout 3% to about 7%, from about 3% to about 6%, from about 3% to about5%, from about 3% to about 4%, from about 4% to about 10%, from about 4%to about 9%, from about 4% to about 8%, from about 4% to about 7%, fromabout 4% to about 6%, from about 4% to about 5%, from about 5% to about10%, from about 5% to about 9%, from about 5% to about 8%, from about 5%to about 7%, from about 5% to about 6%, from about 6% to about 10%, fromabout 6% to about 9%, from about 6% to about 8%, from about 6% to about7%, from about 7% to about 10%, from about 7% to about 9%, from about 7%to about 8%, from about 8% to about 10%, from about 8% to about 9%, orfrom about 9% to about 10% by weight of the total weight of thesolution.

In certain embodiments, the amount of the oxidized dextran compositionis from about 1% (w/w) to about 25% (w/w). In certain embodiments, theamount of the oxidized dextran composition is from about 1% (w/w) toabout 10% (w/w) by weight of the total weight of the solution. Incertain embodiments, the amount of the oxidized dextran composition isfrom about 2% to about 5%, about 4% to about 7%, or about 6% to about9%.

In another aspect, provided herein is a method of preparing an oxidizeddextran composition comprising:

-   -   (a) contacting a raw dextran material with a periodate salt to        form an oxidized dextran intermediate, wherein the raw dextran        material has a PDI of from about 1.4 to about 5.0; and    -   (b) purifying the oxidized dextran intermediate to produce an        oxidized dextran composition of the present invention.

In certain embodiments, the periodate salt is selected from the groupconsisting of sodium periodate or potassium periodate.

In certain embodiments, the yield of oxidized dextran obtained followingstep (c) is from about 75% to about 95%.

In certain embodiments, the method further comprising the step ofprecipitating the oxidized dextran composition to provide a solid. Incertain embodiments, the method further comprising the step oflyophilizing the oxidized dextran composition to provide a solid.

In certain embodiments, the amount of the oxidized dextran compositionis from about 1% (w/w) to about 10% (w/w) by weight of the total weightof the solution.

VI Methods of Making Hemostatic Compositions

The hemostatic hydrogels of the present invention represent athree-dimensional dynamic network that forms with reversible covalentbonds. The dynamic network is modulated by a variety of propertiesincluding, among other things, the properties of the two precursorcompositions of the present invention, namely the acrylated chitosan andoxidized dextran compositions. Without wishing to be bound by theory, itis believed that the formation of the hydrogel composition is a two-stepprocess. In the first step, the acrylated chitosan and oxidized dextranpolymer chains intertwine, resulting in the formation of hydrogenbonding between the acrylated chitosan and oxidized dextran chains. Inthe second step, a Schiff base group forms between the aldehyde groupspresent in the oxidized dextran and the free amino group of theacrylated chitosan. The localized reaction between one monomer of theoxidized dextran (in its aldehyde form) and one monomer of the acrylatedchitosan to link via Schiff base formation is depicted below:

Schiff base formation is a reversible reaction with equilibrium favoringformation of the Schiff base. This reversibility in Schiff baseformation may impact the biodegradation of the hydrogel over time. It iscontemplated more unreacted aldehydes on the oxidized dextran backbonecome in contact with unreacted amines on the acrylated chitosan backboneto form additional Schiff base links, the polymer chains reorient to amore compact hydrogel with a reduced spatial volume that exudes water asit shrinks.

The kinetics of hydrogel formation are important for the function of thehydrogel as a hemostat given, among other things, the desire to rapidlystop blood loss or leakage in tissues of interest. It is believed thatthe hydrogel compositions of the present invention have three differentreaction kinetics occurring concurrently. The initial reaction kineticsof gel formation are considered rapid enough for the hydrogel to adhereto tissue and gain enough cohesive strength and stiffness in a shortperiod of time (e.g., from about 5 to about 30 seconds) to stem theblood flow. During this short period of time, the initial kinetics alsoforms a trabeculated, non-interconnected pore structure with a largepore size distribution (see, FIGS. 1 and 5). These features contributeto the collection, retention and concentration of platelets and redblood cells (RBCs) within the pore structure of the hydrogel. Thesurface roughness of this pore structure facilitates platelet adhesion,activation and aggregation (e.g., soft clot or platelet plug) which arethe first steps in activating the coagulation cascade (see, FIG. 2).Once the coagulation cascade is triggered, fibrin strands are formedthrough biochemical pathways. The fibrin strands trap more platelets andred blood cells, thereby forming a stable blood clot. The secondary ratekinetics of additional Schiff base formation appear to be slower andretain the concentrated platelets and RBCs locally to promotecoagulation. The lack of swelling in the hydrogel avoids dilution of theplatelets and RBCs, as might be observed in other matrices that expand(e.g., gelatin based hemostats, cellulose based hemostats), and resultsin more effective aggregation and concentration that is required forcoagulation. The third rate kinetics relate to the rate of degradationof the hydrogel. This rate is fast enough to allow continuous shrinkageand eventual degradation within a physiologically relevant time frameover days, weeks and months.

The invention provides methods of preparing hemostatic hydrogelcompositions. In various embodiments, the methods generally includecontacting an acrylated chitosan composition disclosed herein with anoxidized dextran composition disclosed herein.

In certain embodiments, the ratio of the viscosity of the acrylatedchitosan composition to the oxidized chitosan composition ranges fromabout 50:1 to about 10,000:1, from about 100:1 to about 10,000:1, fromabout 500:1 to about 10,000:1, from about 1,000:1 to about 10,000:1,from about 5,000:1 to about 10,000:1, from about 50:1 to about 5,000:1,from about 50:1 to about 1,000:1, from about 50:1 to about 500:1, fromabout 50:1 to about 100:1, from about 100:1 to about 5,000:1, from about100:1 to about 1,000:1, from about 100:1 to about 500:1, from about500:1 to about 5,000:1, from about 500:1 to about 1,000:1, or from about1,000:1 to about 5,000:1. In certain embodiments, the ratio of theviscosity of the acrylated chitosan composition to the oxidized chitosancomposition ranges from about 100:1 to about 10,000:1.

In certain embodiments, the ratio of the volume of aCHN solution to thevolume of oDEX solution is about 10:1, about 9:1, about 8:1, about 7:1,about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8,about 1:9, or about 1:10.

In certain embodiments, the resulting hemostatic hydrogel composition isa hemostatic hydrogel composition disclosed herein. However, it isappreciated that the hydrogel achieves its hemostatic properties andblood coagulation properties in the absence of an additional bloodclotting agent that promotes blood clotting or otherwise activates thethe blood clotting cascade (e.g., Factor XIII, Factor IX, Factor X,Factor XI, Factor XII, or thrombin). As a result, in certainembodiments, the oxidized dextran, acrylated chitosan, and thehemostatic hydrogel produced by combining the acrylated chitosan andoxidized dextran are substantially free of one or more exogenously addedblood clotting agents. In this context, the term “substantially free” ofan exogenously added blood clotting agent is understood to mean anamount of the exogenously added agent that is less than is necessary toproduce a blood clot in a standard blood clotting assay.

In certain embodiments, the hemostatic composition further comprises atherapeutic agent. In certain embodiments, the therapeutic agent isselected from the group consisting of a small molecule drug, peptide(e.g., hormone), protein, for example, hormone, growth factor, andantibody. In certain embodiments, the small molecule drug compound isselected from the group comprising an antibiotic, a non-steroidalanti-inflammatory agent, an anti-cancer agent, an antimicrobial.

In certain embodiments, the therapeutic agent can include, withoutlimitation, a polynucleotide (e.g., DNA, RNA), an oligonucleotide, agene therapy agent, a nucleoside analog (e.g., Cytovene, Epivir, Gemzar,Hivid, Rebetron, Videx, Zerit, Zovirax), a polynucleic acid decoy, apeptide (e.g., peptide hormone), a protein (e.g., a therapeuticantibody, e.g., Ig A, Ig G, Ig M, Ig D, and Ig E antibodies), or agrowth factor (e.g., bone morphic growth factor (BMP), angiopoietin,acidic fibroblast growth factor (aFGF), basic fibroblast growth factor(bFGF), vascular endothelial growth factor (vEGF), platelet derivedgrowth factor (PDGF), nerve growth factor (NGF)), an anti-inflammatorydrug (e.g., dexamethasone), an immune suppressive agent, ananti-neoplastic agent, an anti-cancer agent, an anti-cell proliferationagent, a nitric oxide releasing agent, an anti-diabetic drug (e.g.,rosigliatazone), an antibiotic (e.g., Minocycline, Rifampin,Erythromycin, Novobiocin), an anti-microbial agent (e.g., Triclosan,Fusidic acid, Silver acetate, Polymyxin), an analgesic (e.g., codeine,meperidine, morphine, aspirin, acetaminophen, d-propoxyphene), a burncare agent (e.g., silver sulfadiazine, bacitracin, benzocaine) or anycombination thereof. In certain embodiments, the therapeutic agent maybe added to the acrylated chitosan composition and/or the oxidizeddextran composition prior to contacting the acrylated chitosancomposition with the oxidized dextran composition.

In certain embodiments, the hemostatic composition further comprises aplurality of cells, for example, immune cells (for example, geneticallymodified immune cells such as genetically modified B-cells or T-cells,such as CART cells) and stem cells. In certain embodiments, the stemcells are selected from the group comprising mesenchymal stem cells,pancreatic stem cells, pluripotent stem cells, neural stem cells,hematopoietic stem cells or any combination thereof. The cells may beadded to the acrylated chitosan composition and/or the oxidized dextrancomposition prior to contacting the acrylated chitosan composition withthe oxidized dextran composition.

In certain embodiments, the acrylated chitosan composition is contactedwith the oxidized dextran composition in a static mixer, and optionallyor in addition the mixture is aerosolized prior to application totissue. In certain embodiments, the mixture is aerosolized using anair-assisted spray tip or an unassisted spray tip.

In certain embodiments, the gelation time of the hemostatic hydrogelcomposition forms within from about 5 seconds to about 300 seconds, fromabout 5 seconds to about 240 seconds, from about 5 seconds to about 180seconds, from about 5 seconds to about 120 seconds, from about 5 secondsto about 60 seconds, from about 7 seconds to about 300 seconds, fromabout 7 seconds to about 240 seconds, from about 7 seconds to about 180seconds, from about 7 seconds to about 120 seconds, from about 7 secondsto about 60 seconds, from about 10 seconds to about 300 seconds, fromabout 10 seconds to about 240 seconds, from about 10 seconds to about180 seconds, from about 10 seconds to about 120 seconds, or from about10 seconds to about 60 seconds, after contacting the acrylated chitosancomposition with the oxidized dextran composition.

In certain embodiments, the gelation time of the hemostatic hydrogelcomposition forms within from about 10 seconds to about 240 secondsafter contacting the acrylated chitosan composition with the oxidizeddextran composition. In certain embodiments, the gelation time of thehemostatic hydrogel composition forms within from about 10 seconds aftercontacting the acrylated chitosan composition with the oxidized dextrancomposition. In certain embodiments, the gelation time of the hemostatichydrogel composition forms within from about 240 seconds aftercontacting the acrylated chitosan composition with the oxidized dextrancomposition. In certain embodiments, the hemostatic hydrogelcompositions of the present invention with a gelation time of within 240seconds after contacting the acrylated chitosan composition with theoxidized dextran composition may be useful in the treatment of woundsresulting from minimally invasive surgeries (MIS).

VII Properties of Hemostatic Compositions

The hemostatic hydrogels described herein have a variety of structuraland/or structural/functional features.

(i) Structural Features

For example, the hemostatic hydrogel comprise two or more of thefollowing features, which include (i) a total amount of free aldehydegroups of from about 0.1 to about 0.7 moles aldehyde/mole oxidizeddextran, (ii) the hydrogel composition is formed from oxidized dextranand acrylated chitosan, wherein the ratio of primary aldehydes in theoxidized dextran to the amines in the acrylated chitosan is in the rangefrom about 1.0 to about 2.0, and/or the ratio of total aldehydes in inthe oxidized dextran to amines in the acrylated chitosan is from about1.5 to about 3.0, (iii) the ratio of Mw of the acrylated chitosan to theoxidized dextran is from about 2 to about 10, the ratio of Mn of theacrylated chitosan to the oxidized dextran is from about 4 to about 15,the ratio of Mz of the acrylated chitosan to the oxidized dextran isfrom about 2 to about 10, the ratio of PDI (Mw/Mn) of acrylated chitosanto oxidized dextran is from about 0.5 to about 0.8, and/or the ratio ofPDI (Mz/Mw) of acrylated chitosan to oxidized dextran is from about 0.5to about 1.0, (iv) upon formation of the hydrogel composition, thehydrogel composition comprises a bound water content of from about 65%w/w to about 95% w/w, (v) a three-dimensional porous structurecomprising layers of substantially non-interconnected pores having (a) apore size distribution from about 10 μm to about 850 μm in diameter, (b)a platelet adhesive surface, or a combination of (a) and (b), and/or(vi) the hydrogel composition comprises walls disposed between thesubstantially non-interconnected pores, the walls having a wallthickness of from 0.046 μm to 50 μm.

In certain embodiments, the hydrogel composition increases in burststrength over time. For example, in certain embodiments (i) at about 10seconds after the formation of the hydrogel composition, the hydrogelcomposition has a burst strength of greater than 20 mmHg as determinedusing an ASTM F 2392-04 protocol, (ii) at about 2 minutes after theformation of the hydrogel composition, the hydrogel composition has aburst strength of greater than about 35 mmHg as determined using an ASTMF 2392-04 protocol, and/or (iii) at about 5 minutes after the formationof the hydrogel composition, the hydrogel composition has a burststrength of greater than about 70 mmHg as determined using an ASTM F2392-04 protocol. In certain embodiments, the hydrogel composition (i)has an elastic modulus of from about 500 Pa to about 5000 Pa at fromabout 10 seconds to about 80 seconds after the formation of the hydrogelcomposition, (ii) has a compression modulus of from about 3 kPa to about250 kPa, and/or (iii) has an average adhesion strength of from about 1.0N to about 50.0 N, as determined using an ASTM F 2258-05 protocol.

In certain embodiments, the volume of the hydrogel composition (i) whenformed, does not swell upon exposure to a physiological fluid or bodyfluid, and/or (ii) shrinks by less than about 5%, for example, less thanabout 5%, 3%, 2% about 10 minutes after formation when exposed to aphysiological fluid or body fluid.

In certain embodiments, the hydrogel composition is substantiallytransparent when the hydrogel composition has a thickness of 2 mm to 10mm. This permits a medical practitioner (e.g., surgeon) to visualize theunderlying tissue and/or tissue lesion after application of the hemostatto confirm that bleeding has slowed or stopped.

In certain embodiments, the hydrogel composition comprises a totalamount of free aldehyde groups of from about 0.1 to about 0.7, fromabout 0.1 to about 0.6, from about 0.2 to about 0.6, from about 0.3 toabout 0.6, from about 0.4 to about 0.6, from about 0.5 to about 0.6,from about 0.1 to about 0.5, from about 0.1 to about 0.4, from about 0.1to about 0.3, from about 0.1 to about 0.2, from about 0.2 to about 0.5,from about 0.2 to about 0.4, from about 0.2 to about 0.3, from about 0.3to about 0.5, from about 0.3 to about 0.4, or from about 0.4 to about0.5 moles aldehyde/mole oxidized dextran. In certain embodiments, thehydrogel composition comprises a total amount of free aldehyde groups offrom about 0.1 to about 0.7 moles aldehyde/mole oxidized dextran. Incertain embodiments, the hydrogel composition comprises a total amountof free aldehyde groups of from about 0.2 to about 0.5 molesaldehyde/mole oxidized dextran.

In certain embodiments, the ratio of primary aldehydes in the oxidizeddextran to the amines in the acrylated chitosan is in the range fromabout 1.0 to about 2.0, from about 1.2 to about 2.0, from about 1.4 toabout 2.0, from about 1.6 to about 2.0, from about 1.8 to about 2.0,from about 1.0 to about 1.2, from about 1.0 to about 1.4, from about 1.0to about 1.6, from about 1.0 to about 1.8, from about 1.2 to about 1.4,from about 1.2 to about 1.6, from about 1.2 to about 1.8, from about 1.4to about 1.6, from about 1.4 to about 1.8, or from about 1.6 to about1.8. In certain embodiments, the ratio of primary aldehydes in theoxidized dextran to the amines in the acrylated chitosan is in the rangefrom about 1.0 to about 1.8.

In certain embodiments, the ratio of total aldehydes in in the oxidizeddextran to amines in the acrylated chitosan is from about 1.5 to about3.0, from about 2.0 to about 3.0, from about 2.5 to about 3.0, fromabout 1.5 to about 2.5, from about 1.5 to about 2.0, or from about 2.0to about 2.5. In certain embodiments, the ratio of total aldehydes in inthe oxidized dextran to amines in the acrylated chitosan is from about1.5 to about 2.8.

In certain embodiments, the ratio of Mw of the acrylated chitosan to theoxidized dextran is from about 2 to about 10, from about 4 to about 10,from about 6 to about 10, from about 8 to about 10, from about 2 toabout 8, from about 2 to about 6, from about 2 to about 4, from about 4to about 8, from about 4 to about 6, or about from about 6 to about 8.In certain embodiments, the ratio of Mw of the acrylated chitosan to theoxidized dextran is from about 4 to about 8.

In certain embodiments, the ratio of Mn of the acrylated chitosan to theoxidized dextran is from about 4 to about 15, from about 8 to about 15,from about 12 to about 15, from about 4 to about 12, from about 4 toabout 8, or from about 8 to about 12. In certain embodiments, the ratioof Mn of the acrylated chitosan to the oxidized dextran is from about 8to about 15.

In certain embodiments, the ratio of Mz of the acrylated chitosan to theoxidized dextran is from about 2 to about 10, from about 4 to about 10,from about 6 to about 10, from about 8 to about 10, from about 2 toabout 8, from about 2 to about 6, from about 2 to about 4, from about 4to about 8, from about 4 to about 6, or from about 6 to about 8. Incertain embodiments, the ratio of Mz of the acrylated chitosan to theoxidized dextran is from about 4 to about 8.

In certain embodiments, the ratio of PDI (Mw/Mn) of acrylated chitosanto oxidized dextran is from about 0.5 to about 0.8, from about 0.6 toabout 0.8, from about 0.7 to about 0.8, from about 0.5 to about 0.7,from about 0.5 to about 0.6, or from about 0.6 to about 0.7. In certainembodiments, the ratio of PDI (Mw/Mn) of acrylated chitosan to oxidizeddextran is from about 0.5 to about 0.7.

In certain embodiments, the ratio of PDI (Mz/Mw) of acrylated chitosanto oxidized dextran is from about 0.5 to about 1.0, from about 0.6 toabout 1.0, from about 0.7 to about 1.0, from about 0.8 to about 1.0,from about 0.9 to about 1.0, from about 0.5 to about 0.9, from about 0.5to about 0.8, from about 0.5 to about 0.7, from about 0.5 to about 0.6,from about 0.6 to about 0.9, from about 0.6 to about 0.8, from about 0.6to about 0.7, from about 0.7 to about 0.9, from about 0.7 to about 0.8,or from about 0.8 to about 0.9. In certain embodiments, the ratio of PDI(Mz/Mw) of acrylated chitosan to oxidized dextran is from about 0.8 toabout 1.0.

In certain embodiments, upon formation of the hydrogel composition, thehydrogel composition comprises a bound water content of from about 60%w/w to about 99% w/w, from about 65% w/w to about 99% w/w, from about70% w/w to about 99% w/w, from about 75% w/w to about 99% w/w, fromabout 80% w/w to about 99% w/w, from about 85% w/w to about 99% w/w,from about 90% w/w to about 99% w/w, from about 95% w/w to about 99%w/w, from about 60% w/w to about 95% w/w, from about 60% w/w to about90% w/w, from about 60% w/w to about 85% w/w, from about 60% w/w toabout 80% w/w, from about 60% w/w to about 75% w/w, from about 60% w/wto about 70% w/w, from about 60% w/w to about 65% w/w, from about 65%w/w to about 95% w/w, from about 65% w/w to about 90% w/w, from about65% w/w to about 85% w/w, from about 65% w/w to about 80% w/w, fromabout 65% w/w to about 75% w/w, from about 65% w/w to about 70% w/w,from about 70% w/w to about 95% w/w, from about 70% w/w to about 90%w/w, from about 70% w/w to about 85% w/w, from about 70% w/w to about80% w/w, from about 70% w/w to about 75% w/w, from about 75% w/w toabout 95% w/w, from about 75% w/w to about 90% w/w, from about 75% w/wto about 85% w/w, from about 75% w/w to about 80% w/w, from about 80%w/w to about 95% w/w, from about 80% w/w to about 90% w/w, from about80% w/w to about 85% w/w, from about 85% w/w to about 95% w/w, fromabout 85% w/w to about 90% w/w, or from about 90% w/w to about 95% w/w.In certain embodiments, upon formation of the hydrogel composition, thehydrogel composition comprises a bound water content of from about 65%w/w to about 95% w/w.

In certain embodiments, the hemostatic hydrogel composition comprises athree-dimensional porous structure, that comprises layers ofsubstantially non-interconnected pores (see, e.g., FIG. 1). In certainembodiments, the layers of substantially non-interconnected porescomprise anisotropic pores and/or isotropic pores. In certainembodiments, the layers of substantially non-interconnected porescomprise both anisotropic pores and isotropic pores. In certainembodiments, the pores have a pore size distribution from about 10 μm toabout 850 μm in diameter, where the diameter of a pore is the longestpossible straight-line distance between the edges of the pore.

In certain embodiments, the ratio of the pore volume to the hydrogelvolume is from about 0.9 to about 0.98. In certain embodiments, theratio of the volume of the walls disposed between the pores to thehydrogel volume is from about 0.02 to about 0.09. In certainembodiments, the ratio of the pore volume to the volume of the wallsdisposed between the pores is from about 10 to about 33.

In certain embodiments, at about 2 minutes after the formation of thehydrogel composition, the hydrogel composition has a burst strength offrom about 35 mmHg to about 80 mmHg, from about 40 mmHg to about 80mmHg, from about 45 mmHg to about 80 mmHg, from about 50 mmHg to about80 mmHg, from about 60 mmHg to about 80 mmHg, from about 70 mmHg toabout 80 mmHg, from about 35 mmHg to about 70 mmHg, from about 35 mmHgto about 60 mmHg, from about 35 mmHg to about 50 mmHg, from about 35mmHg to about 45 mmHg, from about 35 mmHg to about 40 mmHg, from about40 mmHg to about 80 mmHg, from about 40 mmHg to about 70 mmHg, fromabout 40 mmHg to about 60 mmHg, from about 40 mmHg to about 50 mmHg,from about 40 mmHg to about 45 mmHg, from about 50 mmHg to about 80mmHg, from about 50 mmHg to about 70 mmHg, from about 50 mmHg to about60 mmHg, from about 60 mmHg to about 80 mmHg, from about 60 mmHg toabout 70 mmHg, or from about 70 mmHg to about 80 mmHg, as determinedusing an ASTM F 2392-04 protocol.

In certain embodiments, at about 5 minutes after the formation of thehydrogel composition, the hydrogel composition has a burst strength offrom about 70 mmHg to about 125 mmHg, from about 70 mmHg to about 115mmHg, from about 70 mmHg to about 110 mmHg, from about 75 mmHg to about110 mmHg, from about 80 mmHg to about 110 mmHg, from about 85 mmHg toabout 110 mmHg, from about 90 mmHg to about 110 mmHg, from about 100mmHg to about 110 mmHg, from about 70 mmHg to about 100 mmHg, from about70 mmHg to about 90 mmHg, from about 70 mmHg to about 85 mmHg, fromabout 70 mmHg to about 80 mmHg, from about 70 mmHg to about 75 mmHg,from about 75 mmHg to about 100 mmHg, from about 75 mmHg to about 90mmHg, from about 75 mmHg to about 85 mmHg, from about 75 mmHg to about80 mmHg, from about 80 mmHg to about 100 mmHg, from about 80 mmHg toabout 90 mmHg, from about 80 mmHg to about 85 mmHg, from about 85 mmHgto about 100 mmHg, from about 85 mmHg to about 90 mmHg, or from about 90mmHg to about 100 mmHg, as determined using an ASTM F 2392-04 protocol.

In certain embodiments, the hemostatic hydrogel composition, whenformed, does not swell upon exposure to a physiological fluid or bodyfluid.

In certain embodiments, the volume of the hydrogel composition shrinksby less than about 1.0%, about 1.2%, about 1.4%, about 1.6%, about 1.8%,about 2.0%, about 2.2%, about 2.4%, about 2.6%, about 2.8%, about 3.0%,about 3.2%, about 3.4%, about 3.6%, about 3.8%, about 4.0%, about 4.2%,about 4.4%, about 4.6%, about 4.8%, or about 5%, about 10 minutes afterformation when exposed to a physiological fluid or body fluid.

In certain embodiments, the hydrogel composition is substantiallytransparent when the hydrogel composition has a thickness of 2 mm to 10mm. In certain embodiments, a substantially transparent hydrogelcomposition maybe defined as any hydrogel composition with a visiblelight % transmittance of greater than 65%, wherein the % transmittanceof the hydrogel composition is measured using a UV-vis spectrometer.

In certain embodiments, the hemostatic hydrogel comprises pores certainof which having platelet adhesive surfaces that are sufficiently roughto permit activated platelets and/or red blood cells to adhere to theplatelet adhesive surfaces.

In certain embodiments, the hemostatic hydrogel compositions of thepresent invention comprise:

-   -   (a) from about 0.0 to about 0.3 mole fraction of a first monomer        of formula (I)

-   -   (b) from about 0.02 to about 0.7 mole fraction of a second        monomer of formula (III),

and

-   -   (c) about 0.0 to about 0.8 mole fraction of a second monomer of        formula (V),

In certain embodiments, the hemostatic hydrogel composition comprisesfrom about 0.0 to about 0.26, from about 0.05 to about 0.26, from about0.1 to about 0.26, from about 0.15 to about 0.26, from about 0.2 toabout 0.26, from about 0.0 to about 0.2, from about 0.0 to about 0.15,from about 0.0 to about 0.1, from about 0.0 to about 0.05, from about0.05 to about 0.2, from about 0.05 to about 0.15, from about 0.05 toabout 0.1, from about 0.1 to about 0.2, from about 0.1 to about 0.15, orfrom about 0.15 to about 0.2, mole fraction of the first monomer offormula (I). In certain embodiments, the hemostatic hydrogel compositioncomprises from about 0.0 to about 0.26 mole fraction of the firstmonomer of formula (I).

In certain embodiments, the hemostatic hydrogel composition comprisesfrom about 0.03 to about 0.7, from about 0.03 to about 0.6, from about0.05 to about 0.6, from about 0.1 to about 0.6, from about 0.3 to about0.6, from about 0.5 to about 0.6, from about 0.03 to about 0.5, fromabout 0.03 to about 0.3, from about 0.03 to about 0.1, from about 0.03to about 0.05, from about 0.05 to about 0.5, from about 0.05 to about0.3, from about 0.05 to about 0.1, from about 0.1 to about 0.5, fromabout 0.1 to about 0.3, or from about 0.3 to about 0.5, mole fraction ofthe third monomer of formula (III). In certain embodiments, wherein thehemostatic hydrogel composition from about 0.03 to about 0.6 molefraction of the third monomer of formula (III).

In certain embodiments, the hemostatic hydrogel composition comprisesfrom about 0.02 to about 0.8, from about 0.04 to about 0.8, from about0.0 to about 0.7, from about 0.02 to about 0.7, from about 0.04 to about0.7, from about 0.1 to about 0.7, from about 0.3 to about 0.7, fromabout 0.5 to about 0.7, from about 0.04 to about 0.5, from about 0.04 toabout 0.3, from about 0.04 to about 0.1, from about 0.1 to about 0.5,from about 0.1 to about 0.3, or from about 0.3 to about 0.5, molefraction of the first monomer of formula (V). In certain embodiments,the hemostatic hydrogel composition comprises from about 0.04 to about0.7 mole fraction of the first monomer of formula (V).

In certain embodiments, wherein the hemostatic hydrogel compositioncomprises no greater than about 0.2 mole fraction of a fourth monomer offormula (IV)

In certain embodiments, the hemostatic hydrogel composition furthercomprises from about 0.0 to about 0.2, from about 0.05 to about 0.2,from about 0.1 to about 0.2, from about 0.15 to about 0.2, from about0.0 to about 0.15, from about 0.0 to about 0.1, from about 0.0 to about0.05, from about 0.05 to about 0.15, from about 0.05 to about 0.1, orfrom about 0.1 to about 0.15, mole fraction of the fourth monomer offormula (IV).

In certain embodiments, the hemostatic hydrogel composition comprises nogreater than about 0.65 mole fraction of a fifth monomer of formula(VIII)

In certain embodiments, the hemostatic hydrogel composition comprisesfrom about 0.0 to about 0.65, from about 0.2 to about 0.65, from about0.4 to about 0.65, from about 0.0 to about 0.4, from about 0.0 to about0.2, or from about 0.2 to about 0.4, mole fraction of a third monomer offormula (VIII).

In certain embodiments, the hydrogel comprises a plurality ofcrosslinked moieties of formula (IX)

(ii) Structural/Functional Features

In certain embodiments, the hydrogels contain pores certain of whichhave a platelet adhesive surface so that, when in contact with blood,the hydrogel composition permits (i) platelets and/or red blood cells tobind to the platelet adhesive surface and promote blood clot formationat or within the hydrogel composition, and/or (ii) platelets and/or redblood cells to adhere to the platelet adhesive surface and not permitplatelets and/or red blood cells from the blood to enter pores presentin a first surface of the hydrogel composition, pass through thehydrogel composition, and then exit the hydrogel composition via porespresent in a second surface of the hydrogel composition that opposes thefirst surface.

The invention provides methods of promoting hemostasis (e.g., reducingor stopping blood loss) at a location in a subject in need thereof byapplying a hydrogel composition of the invention to the location to betreated in the subject.

In certain embodiments, the hemostatic hydrogel composition adheres to atissue surface at the location. In certain embodiments, the hemostatichydrogel composition adheres to the tissue surface at the location withaverage adhesion strength of from about 1.0 N to about 50.0 N, asdetermined using an ASTM F 2258-05 protocol.

In certain embodiments, platelets and/or red blood cells present at thelocation adhere to surfaces of pores in the hemostatic hydrogel.

In certain embodiments, the hemostatic hydrogel composition promotesplatelet adhesion, platelet activation, and platelet aggregation at thesite in need of hemostasis. During use, the hemostatic hydrogelcomposition promotes a blood coagulation cascade and blood clotformation at the site.

In certain embodiments, blood coagulation is achieved from about 10seconds to about 300 seconds, from about 20 seconds to about 300seconds, from about 30 seconds to about 300 seconds, from about 60seconds to about 300 seconds, from about 120 seconds to about 300seconds, from about 180 seconds to about 300 seconds, from about 240seconds to about 300 seconds, from about 10 seconds to about 240seconds, from about 10 seconds to about 180 seconds, from about 10seconds to about 120 seconds, from about 10 seconds to about 60 seconds,from about 10 seconds to about 30 seconds, from about 10 seconds toabout 20 seconds, from about 20 seconds to about 240 seconds, from about20 seconds to about 180 seconds, from about 20 seconds to about 120seconds, from about 20 seconds to about 60 seconds, from about 20seconds to about 30 seconds, from about 30 seconds to about 240 seconds,from about 30 seconds to about 180 seconds, from about 30 seconds toabout 120 seconds, from about 30 seconds to about 60 seconds, from about60 seconds to about 240 seconds, from about 60 seconds to about 180seconds, from about 60 seconds to about 120 seconds, from about 120seconds to about 240 seconds, from about 120 seconds to about 180seconds, or from about 180 seconds to about 240 seconds. In certainembodiments, when a hemostatic hydrogel composition of the presentinvention is applied to an abrasion, blood coagulation is achieved fromabout 20 seconds to about 210 seconds.

In certain embodiments, the hemostatic hydrogel compositions degradewithin days, weeks (e.g., in 1 week, 2 weeks, 3 weeks, or 4 weeks) ormonths (e.g., two, three, four, five or six months).

In certain embodiments, the hemostatic hydrogel compositions of thepresent invention are biodegradable. Without wishing to be bound bytheory it is believed that degradation of the hydrogels occurs byhydrolysis and enzymatic degradation. The degradation pathway may occurthrough a combination of two mechanisms. A first mechanism may involvecleavage of the crosslinks (e.g., the Schiff base linkages) between thepolymer chains producing water soluble fragments. It is believed thatthe Schiff base linkages are reversible which leads to a progressivereorganization during which the polymer chains become degraded. A secondmechanism may involve the degradation of the acrylated chitosan andoxidized dextran chains to produce smaller fragments (e.g., oligomers ormonomers that are water soluble). Given that the hydrogel is formed fromnaturally occurring sugars, the products of degradation are non-toxicand are further degraded by the carbohydrate metabolism pathways orrapidly eliminated by the renal system.

It is believed that one of the rate limiting steps for the degradationprocess is the degree of acetylation (DA) of the acrylated chitosan.Given that certain enzymes, e.g., lysozyme, require a linear series ofat least three N-acetylglucosamine units to cleave a glycidic link. Manyenzymes have a hydrophobic pocket that interact with the polymer e.g.,around three linear N-acetylglucosamine subunits, and catalyzes thechain cleavage. The probability of having three N-acetylglucosamineunits in a row is given by DA×DA×DA=DA³. As a result, as DA decreases,the probability of having three linear, adjacent N-acetylglucosaminesubunits together drops as the third power DA. It is understood,however, that the rate of degradation can be modified or adjusted byaltering the DA content of acrylated chitosan used to create thehydrogel.

In another aspect, provided is a hemostatic hydrogel compositioncomprising two or more of the following features:

-   -   (i) the hydrogel composition comprises a three-dimensional        porous structure comprising layers of substantially        non-interconnected pores having (a) a pore size distribution        from about 10 μm to about 850 μm in diameter, (b) a platelet        adhesive surface, or a combination of (a) and (b),    -   (ii) the hydrogel composition comprises walls disposed between        the substantially non-interconnected pores, the walls having a        wall thickness of from 0.046 μm to 50 μm,    -   (iii) the hydrogel composition comprises a platelet adhesive        surface so that, when in contact with blood, the hydrogel        composition permits platelets and/or red blood cells within the        blood to adhere to platelet adhesive surface and promote blood        clot formation at or within the hydrogel composition,    -   (iv) the hydrogel composition comprises a platelet adhesive        surface so that, when in contact with blood, the hydrogel        composition permits platelet and/or red blood cells within the        blood to adhere to the platelet adhesive surface and not permit        platelets and/or red blood cells from the blood to enter pores        present in a first surface of the hydrogel composition, pass        through the hydrogel composition, and then exit the hydrogel        composition via pores present in a second surface of the        hydrogel composition that opposes the first surface,    -   (v) at about 10 seconds after the formation of the hydrogel        composition, the hydrogel composition has a burst strength of        greater than 20 mmHg as determined using an ASTM F 2392-04        protocol,    -   (vi) at about 2 minutes after the formation of the hydrogel        composition, the hydrogel composition has a burst strength of        greater than about 35 mmHg as determined using an ASTM F 2392-04        protocol,    -   (vii) at about 5 minutes after the formation of the hydrogel        composition, the hydrogel composition has a burst strength of        greater than about 70 mmHg as determined using an ASTM F 2392-04        protocol,    -   (viii) the hydrogel composition has an elastic modulus of from        about 500 Pa to about 5000 Pa at from about 10 seconds to about        80 seconds after the formation of the hydrogel composition,    -   (ix) the hydrogel composition has a compression modulus of from        about 3 kPa to about 250 kPa,    -   (x) the hydrogel composition has an average adhesion strength of        from about 1.0 N to about 50.0 N, as determined using an ASTM F        2258-05 protocol,    -   (xi) the volume of the hydrogel composition, when formed, does        not increase upon exposure to a physiological fluid or body        fluid,    -   (xii) the volume of the hydrogel composition shrinks by less        than about 5% about 10 minutes after formation when exposed to a        physiological fluid or body fluid,    -   (xiii) the hydrogel composition is substantially transparent        when the hydrogel composition has a thickness of 2 mm to 10 mm,        and    -   (xiv) the hydrogel composition optionally further comprises a        therapeutic agent.

VIII Methods of Treatment and Administration

It is understood that the hemostatic hydrogel compositions can be usedto promote hemostasis in a variety of scenarios, for example, when theblood loss is caused by trauma, abrasion, or surgical or other medicalintervention at the location.

In certain embodiments, the surgical intervention is selected from anarthroscopic procedure, a cardiac surgery, a cranial surgery, anendoscopic procedure, a laparoscopic procedure, an OB-GYN surgery, anorgan surgery, a plastic surgery, a sinus procedure, a spinal surgery, athoracic surgery, or a vascular surgery. In certain circumstances, thecranial surgery can comprise a procedure to remove a hematoma or atumor. In certain embodiments, the surgical intervention is a minimallyinvasive surgery, an endoscopic procedure, a laparoscopic procedure, oran arthroscopic procedure. In other approaches, the hemostatic hydrogelcompositions of the present invention can be used as a topical wounddressing, for example, for the treatment of burns.

It is understood that, when appropriate, the hemostatic hydrogelcomposition, when applied to the site, is capable of filling a cavity atthe site without inducing compression of tissue surrounding the cavitywhen the hemostatic composition is exposed to physiological fluid or abody fluid. This can be particularly important during brain or spinalsurgery where swelling of the hemostat in these locations is undesirableas it can cause compression of surrounding tissue.

In addition, it is understood that the hemostatic hydrogel can be usedto facilitate the delivery of a therapeutic agent to a site if intent,for example, during surgery. In certain embodiments, the therapeuticagent is selected from the group consisting of a small molecule drug, agrowth factor, a hormone, an antibody, an anti-cancer agent, anantimicrobial. In certain embodiments, the small molecule drug compoundis selected from the group comprising an antibiotic, a non-steroidalanti-inflammatory agent.

In certain embodiments, the therapeutic agent can include, withoutlimitation, a polynucleotide (e.g., DNA, RNA), an oligonucleotide, agene therapy agent, a nucleoside analog (e.g., Cytovene, Epivir, Gemzar,Hivid, Rebetron, Videx, Zerit, Zovirax), a polynucleic acid decoy, apeptide (e.g., peptide hormone), a protein (e.g., a therapeuticantibody, e.g., Ig A, Ig G, Ig M, Ig D, and Ig E antibodies), or agrowth factor (e.g., bone morphic growth factor (BMP), angiopoietin,acidic fibroblast growth factor (aFGF), basic fibroblast growth factor(bFGF), vascular endothelial growth factor (vEGF), platelet derivedgrowth factor (PDGF), nerve growth factor (NGF)), an anti-inflammatorydrug (e.g., dexamethasone), an immune suppressive agent, ananti-neoplastic agent, an anti-cancer agent, an anti-cell proliferationagent, a nitric oxide releasing agent, an anti-diabetic drug (e.g.,rosigliatazone), an antibiotic (e.g., Minocycline, Rifampin,Erythromycin, Novobiocin), an anti-microbial agent (e.g., Triclosan,Fusidic acid, Silver acetate, Polymyxin), an analgesic (e.g., codeine,meperidine, morphine, aspirin, acetaminophen, d-propoxyphene), a burncare agent (e.g., silver sulfadiazine, bacitracin, benzocaine) or anycombination thereof.

In certain embodiments, the method further comprising administering aplurality of cells, for example, immune cells, e.g., engineered immunecells (e.g., CAR T cells) or stem cells to the site. In certainembodiments, the hemostatic composition comprises a plurality of stemcells. In certain embodiments, the plurality of stem cells comprise stemcells selected from mesenchymal stem cells, pancreatic stem cells,pluripotent stem cells, neural stem cells, hematopoietic stem cells orany combination thereof. In certain embodiments, the hemostatic hydrogelcompositions of the present invention can be used to promote tissueregeneration.

In certain embodiments, when a hemostatic hydrogel composition of thepresent invention is applied to an abrasion, hemostasis is achieved, forexample, from about 5 seconds to about 120 seconds, from about 10seconds to about 120 seconds, from about 15 seconds to about 120seconds, from about 5 seconds to about 60 seconds, from about 10 secondsto about 60 seconds, from about 15 seconds to about 60 seconds, fromabout 5 seconds to about 30 seconds, from about 10 seconds to about 30seconds, from about 15 seconds to about 30 seconds, from about 5 secondsto about 15 seconds, or from about 10 seconds to about 15 seconds, afterthe hemostatic hydrogel composition is applied to the location. Incertain embodiments, when a hemostatic hydrogel composition of thepresent invention is applied to an abrasion, hemostasis is achieved fromabout 10 seconds to about 40 seconds, after the hemostatic hydrogelcomposition is applied to the location.

In certain embodiments, when a hemostatic hydrogel composition of thepresent invention is applied to a biopsy punch, hemostasis is achieved,for example, from about 5 seconds to about 60 seconds, from about 10seconds to about 60 seconds, from about 15 seconds to about 60 seconds,from about 20 seconds to about 60 seconds, from about 25 seconds toabout 60 seconds, from about 30 seconds to about 60 seconds, from about40 seconds to about 60 seconds, from about 50 seconds to about 60seconds, from about 5 seconds to about 50 seconds, from about 5 secondsto about 40 seconds, from about 5 seconds to about 35 seconds, fromabout 5 seconds to about 30 seconds, from about 5 seconds to about 25seconds, from about 5 seconds to about 20 seconds, from about 5 secondsto about 15 seconds, from about 5 seconds to about 10 seconds, fromabout 10 seconds to about 50 seconds, from about 10 seconds to about 40seconds, from about 10 seconds to about 35 seconds, from about 10seconds to about 30 seconds, from about 10 seconds to about 25 seconds,from about 10 seconds to about 20 seconds, from about 10 seconds toabout 15 seconds, from about 15 seconds to about 50 seconds, from about15 seconds to about 40 seconds, from about 15 seconds to about 35seconds, from about 15 seconds to about 30 seconds, from about 15seconds to about 25 seconds, from about 15 seconds to about 20 seconds,from about 20 seconds to about 50 seconds, from about 20 seconds toabout 40 seconds, from about 20 seconds to about 35 seconds, from about20 seconds to about 30 seconds, from about 20 seconds to about 25seconds, from about 25 seconds to about 50 seconds, from about 25seconds to about 40 seconds, from about 25 seconds to about 35 seconds,from about 25 seconds to about 30 seconds, from about 30 seconds toabout 50 seconds, from about 30 seconds to about 40 seconds, from about30 seconds to about 35 seconds, from about 35 seconds to about 50seconds, from about 35 seconds to about 40 seconds, or from about 35seconds to about 40 seconds, after the hemostatic hydrogel compositionis applied to the location. In certain embodiments, when a hemostatichydrogel composition of the present invention is applied to a biopsypunch, hemostasis is achieved from about 10 seconds to about 20 seconds,after the hemostatic hydrogel composition is applied to the location.

In certain embodiments, when a hemostatic hydrogel composition of thepresent invention is applied to a laceration, hemostasis is achieved,for example, from about 10 seconds to about 100 seconds, from about 20seconds to about 100 seconds, from about 30 seconds to about 100seconds, from about 40 seconds to about 100 seconds, from about 50seconds to about 100 seconds, from about 70 seconds to about 100seconds, from about 90 seconds to about 100 seconds, from about 10seconds to about 90 seconds, from about 10 seconds to about 70 seconds,from about 10 seconds to about 50 seconds, from about 10 seconds toabout 40 seconds, from about 10 seconds to about 30 seconds, from about10 seconds to about 20 seconds, from about 20 seconds to about 90seconds, from about 20 seconds to about 70 seconds, from about 20seconds to about 50 seconds, from about 20 seconds to about 40 seconds,from about 20 seconds to about 30 seconds, from about 30 seconds toabout 90 seconds, from about 30 seconds to about 70 seconds, from about30 seconds to about 50 seconds, from about 30 seconds to about 40seconds, from about 40 seconds to about 90 seconds, from about 40seconds to about 70 seconds, from about 40 seconds to about 50 seconds,from about 50 seconds to about 90 seconds, from about 50 seconds toabout 70 seconds, or from about 70 seconds to about 90 seconds, afterthe hemostatic hydrogel composition is applied to the location.

In certain embodiments, when a hemostatic hydrogel composition of thepresent invention is applied to a laceration, hemostasis is achieved,for example, from about 10 seconds to about 90 seconds, after thehemostatic hydrogel composition is applied to the location. In certainembodiments, when a hemostatic hydrogel composition of the presentinvention is applied to a laceration, hemostasis is achieved from about80 seconds to about 180 seconds, after the hemostatic hydrogelcomposition is applied to the location.

In certain embodiments, the hemostatic hydrogel composition is appliedto the location in the form of a spray. In certain embodiments, thehemostatic hydrogel composition is applied to the location in the formof a flowable gel. In certain embodiments, the hemostatic hydrogelcomposition is applied to the location in the form of a liquid bandage.

Kits

In various embodiments, the invention provides kits for producinghemostasis (e.g., reducing or stopping blood loss) at a location in asubject in need thereof. In certain embodiments, a kit comprises: i)instructions for producing hemostasis at a location in a subject in needthereof, ii) an acrylated chitosan composition described herein, and/oriii) an oxidized dextran composition described herein, and an optionalmixing system for mixing the acrylated chitosan and oxidized dextran andan optional dispensing system for dispensing a mixture of the acrylatedchitosan and the oxidized dextran.

In certain embodiments, the kit comprises a container (for example,syringe, tube, or bottle) containing the acrylated chitosan compositiondescribed herein and a container (for example, syringe, tube or bottle)containing the oxidized dextran composition described herein inrespective amounts sufficient to produce a hemostatic hydrogel forfacilitating hemostasis at the location in the subject in need thereof.In certain embodiments, the kit comprises on or more containers of theacrylated chitosan composition and the oxidized dextran compositiondescribed herein in respective amounts sufficient to produce ahemostatic hydrogel for facilitating hemostasis at one or more locationsin the subject in need thereof. In certain embodiments, the kitcomprises the acrylated chitosan composition and the oxidized dextrancomposition described herein in amounts sufficient to produce hemostasisat 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 locations in the subject in needthereof.

EXAMPLES

The invention now being generally described, will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1: Method of Making an Exemplary Acrylated Chitosan Composition

This example describes a method for making an acrylated chitosan for usein creating a hemostatic composition.

An exemplary acrylated chitosan was prepared as follows:

Step 1—Preparation of a Chitosan Intermediate

4.0 g of raw chitosan (Mw=162 kDa, PDI=2.3, DDA=79%) starting materialwas dissolved in 200 g of 1% (v/v) acetic acid solution for 30 minutesat 25° C. to produce a mixture. The mixture was then heated to 100° C.and held at this temperature for 2 hours.

Step 2—Preparation of the Acrylated Chitosan Intermediate

The mixture comprising the chitosan intermediate was then cooled to 50°C. Thereafter 5.3 g of acrylic acid was added and the mixture was thenheated to 100° C. and held at this temperature for 60 minutes. Thereaction was then cooled to 28° C. and then quenched by the addition ofNaOH until a final pH value of 12.4 was achieved.

Step 3—Preparation of the Acrylated Chitosan Composition

The mixture comprising the acrylated chitosan intermediate was thenpurified by dialysis for 8 hours to remove salts and low molecularweight components from the mixture to achieve a pH value of 9.1. Thepurified acrylated chitosan was recovered by precipitation fromisopropanol which resulted in a yield of 88%.

Finally, the material was characterized using 1H-NMR spectroscopy andwas determined that the mole fraction of the monomer of formula (III)was 0.37.

Example 2: Method of Making an Exemplary Oxidized Dextran Composition

This example describes a method for making an oxidized dextran for usein creating a hemostatic composition.

An exemplary oxidized dextran was prepared as follows:

Step 1—Preparation of the Oxidized Dextran Intermediate

A dextran solution was prepared by dissolving 5.0 g of raw dextranstarting material (Mw=129 kDa, PDI=4.58) in 400 g of distilled water for30 minutes at 25° C. Thereafter a sodium periodate solution was preparedby dissolving 3.28 g of sodium periodate in 50 g of distilled water. Thesodium periodate solution was then added to the dextran solution and theresulting mixture was stirred for 24 hours.

Step 2—Preparation of the Oxidized Dextran Composition

The mixture comprising the oxidized dextran intermediate was thenpurified by dialysis for 8 hours in order to remove salts and lowmolecular weight components from the mixture. The purified oxidizeddextran was recovered by precipitation from ethanol which resulted in ayield of 88%.

The total amount of aldehyde groups present on the oxidized dextran wasmeasured using the following titration method:

150 mg of oxidized dextran was added to 35 ml of hydroxylaminehydrochloride solution and allowed to stir for 24 hours at roomtemperature. This solution was titrated with a NaOH solution. Thetitration was followed potentiometrically, with the pH recorded as afunction NaOH volume. The volume of NaOH solution used to titrate theoxidized dextran solution was taken to be the endpoint, which wasdetermined as the inflection point of the titrimetric curve.

The total amount of aldehyde groups present on the oxidized dextran wasfound to be 0.76 mol. aldehyde/mol. dextran.

Example 3: Method of Making an Exemplary Hemostatic Composition

This example describes a method for making a hemostatic composition foruse in reducing/stopping bleeding at a tissue lesion.

An exemplary hemostatic composition was prepared as follows:

The acrylated chitosan of Example 1 and oxidized dextran of Example 2were each solubilized into a buffered saline solution to give a finalconcentration of 7.5% (w/w) and 7.5% (w/w), respectively. Equal amountsof the aqueous solution of the oxidized dextran provided in Example 2(concentration: 7.5% w/w) and the aqueous solution of acrylated chitosanprovided in Example 1 (concentration: 7.5% w/w) were loaded in a dualbarrel syringe. A mixer was attached to the top of the dual barrelsyringe. The two solutions were then passed through the mixersimultaneously. The hydrogel was observed to form instantaneously at thetip of the mixer. The combined solution was further aerosolized underpressure.

The burst strength of the acrylated chitosan/oxidized dextran hydrogelwas measured using ASTM F 2392-04 and found to be 96±11 mmHg (C.I of95%).

Example 4: Characterization of the Structural Properties of an ExemplaryHemostatic Hydrogel

This example describes the characterization of the structural propertyin an exemplary hemostatic hydrogel.

An acrylated chitosan composition was produced essentially as describedin Example 1 and an oxidized dextran composition was preparedessentially as described in Example 2. When combined essentially asdescribed in Example 3, the structural property was analyzed usingScanning Electron Microscopy (SEM).

The structure of the acrylated chitosan/oxidized dextran hydrogels wereanalyzed using SEM (see, FIG. 1). The hydrogels were found to haveformed a three-dimensional layered structure comprising, substantiallynon-interconnected anisotropic cells/pores formed between the hydrogelbackbone walls. The layers of cells/pores were staggered andsubstantially non-inter connected with layers directly above and below.As a result, the hydrogels could capture cells (e.g., red blood cells)and prevent them from passing through and exiting the hydrogelstructure.

Example 5: Characterization of the Elastic Modulus of an ExemplaryHemostatic Hydrogel

This example describes the characterization of the elastic modulus in anexemplary hemostatic hydrogel.

An acrylated chitosan composition was produced essentially as describedin Example 1 and an oxidized dextran composition was preparedessentially as described in Example 2. When combined essentially asdescribed in Example 3, the elastic modulus was measured using arheometer. The results are presented in FIG. 3.

The data in FIG. 3 shows that the acrylated chitosan/oxidized dextranhydrogel achieved a desired stiffness of greater than 500 Pa thatincreases over time; which is required for the hemostatic hydrogel toexert an initial tamponade effect when applied to a wound.

Example 6: Characterization of the Gelation Time of an ExemplaryHemostatic Hydrogel

This example describes the characterization of the gelation time of anexemplary hemostatic hydrogel.

An acrylated chitosan composition was produced essentially as describedin Example 1 and an oxidized dextran composition was preparedessentially as described in Example 2. When combined essentially asdescribed in Example 3, the gelation time was measured using stir barstop test. The gelation time was found to be 8 seconds. This exampleshows that the exemplary hemostatic hydrogel forms quickly, which isimportant in achieving fast hemostasis.

Example 7: Characterization of the Rate of Shrinkage of an ExemplaryHemostatic Hydrogel

This example describes the characterization of the rate of shrinkage ofan exemplary hemostatic hydrogel.

An acrylated chitosan composition was produced essentially as describedin Example 1 and an oxidized dextran composition was preparedessentially as described in Example 2. When combined essentially asdescribed in Example 3, the rate of shrinkage was measured as functionof weight loss over time. The rate of shrinkage of the hydrogel is shownin FIG. 4, which demonstrates that the hydrogel shrinks, rather thanswells, overtime. This feature is particularly helpful when a hemostatis used to stop bleeding in confined spaces (e.g., a cranium or a spine)without compressing the nearby tissue.

Example 8: Characterization of the Adhesion Strength of an ExemplaryHemostatic Hydrogel

This example describes the characterization of the adhesion in anexemplary hemostatic hydrogel.

An acrylated chitosan composition was produced essentially as describedin Example 1 and an oxidized dextran composition was preparedessentially as described in Example 2. When combined essentially asdescribed in Example 3, the adhesion strength was measured using ASTM F2392-04 and found to be in the range from 16 to 27N. This example showsthat the exemplary hemostatic hydrogel has enough strength to adhere totissue and function as a hemostat.

Example 9: Method of Reducing/Stopping Bleeding Using an ExemplaryHemostatic Composition

This example describes a method for reducing/stopping bleeding using anexemplary hemostatic composition. In the following studies, theexemplary hemostat was created essentially as described in Example 3,using the acrylated chitosan and oxidized dextran prepared asessentially described in Examples 1 and 2, respectively.

a) High Flow, Low Pressure Model—Liver (Abrasion Wounds)

In this example, the creation of wounds in a porcine liver was used astandard model to evaluate the effect of the acrylated chitosan/oxidizeddextran hydrogel on hemostasis. A Teflon template with a 2 cm diametercircular cut-out to standardize the size of the abrasion wounds. Thewound was created by swiping a Bovie scratch pad 5 times over the liverexposed in the template cut-out. The Bovie scratch pad created ableeding effect similar to that found in human brain surgery. Thebleeding wound was then blotted with dry gauze before the acrylatedchitosan/oxidized dextran hydrogel was applied to the wound.

The transparent hydrogel adhered to the liver tissue and stopped bloodflow immediately. The time to hemostasis was found to be 19±6 seconds(C.I of 95%). Complete coagulation (formation of a clot) occurred in103±36 seconds (C.I of 95%). The formation of a clot was later verifiedusing SEM (see, FIG. 5). FIG. 5 shows the aggregation of activatedplatelets within a pore. Dendritic pseudopodia of multiple plateletswere observed, demonstrating platelet activation.

b) High Flow, High Pressure Model—Liver (Biopsy Punch)

A 10 mm diameter biopsy punch was plunged into a porcine liver, creatinga circular wound, 10 mm in diameter and 5-10 mm in depth. Bleeding wasobserved to occur immediately. The tissue plug created by the punch wasremoved with forceps to create an open wound with high volume, highpressure bleeding. The wound was blotted with dry gauze, and theacrylated chitosan/oxidized dextran hydrogel was filled into the woundcavity, as well as being applied over the wound surface.

The transparent hydrogel adhered to the tissue and stopped blood flowimmediately. The time to hemostasis was found to be between 14 to 18seconds. The formation of a clot was later verified using SEM (see, FIG.6).

c) High Flow, High Pressure Model—Liver (Laceration)

A scalpel was used to create lacerations in a porcine liver, theapproximate dimensions of the lacerations were 30 mm long×16 mm deep.High flow rate, high pressure bleeding was observed to occurimmediately. The wound area was blotted with dry gauze to remove excessblood and allow for visibility. The acrylated chitosan/oxidized dextranhydrogel was filled into the wound cavity, as well as being applied overthe wound surface.

The transparent hydrogel adhered to the tissue and stopped blood flowimmediately. The time to hemostasis was found to be 12 seconds. Theformation of a clot was later verified using SEM (see, FIG. 7).

d) High Flow, High Pressure Model—Spleen (Laceration)

The spleen was used as a model to evaluate the effect of the acrylatedchitosan/oxidized dextran hydrogel on hemostasis as the spleen is a wellvascularized organ that can bleed profusely. A scalpel was used tolacerate the spleen, resulting in lacerations that were 30 mm long×16 mmdeep. The wound was then blotted with dry gauze to remove excess bloodand allow for visibility. The acrylated chitosan/oxidized dextranhydrogel was filled into the wound cavity, as well as applied over thewound surface.

The organ was picked up and observed visually to determine that bloodflow had stopped and that hemostasis had been achieved. The time tohemostasis was found to be 82 seconds. The formation of a clot was laterverified using SEM (see, FIG. 8).

FIG. 8 shows a thick network of fibrin strands trapping multipleactivated platelets and red blood cells. Dendritic pseudopodia ofmultiple platelets was observed, demonstrating platelet activation.

INCORPORATION BY REFERENCE

The entire disclosure of each of the patent documents and scientificarticles referred to herein is incorporated by reference for allpurposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

We claim:
 1. A hemostatic hydrogel composition comprising a gel producedby combining oxidized dextran with an acrylated chitosan compositionproduced by dissolving chitosan in an acetic acid solution prior toacrylation, the hydrogel composition comprising a burst strength ofgreater than about 70 mmHg as determined using an ASTM F 2392-04protocol at about 5 minutes after formation.
 2. The hemostatic hydrogelcomposition of claim 1, wherein the hemostatic hydrogel compositionfurther comprises a total amount of free aldehyde groups of from about0.1 to about 0.7 moles aldehyde/mole oxidized dextran.
 3. The hemostatichydrogel composition of claim 1, wherein the hemostatic hydrogelcomposition further comprises a ratio of primary aldehydes in theoxidized dextran to the amines in the acrylated chitosan from about 1.0to about 2.0, a ratio of total aldehydes in the oxidized dextran toamines in the acrylated chitosan from about 1.5 to about 3.0 or acombination thereof.
 4. The hemostatic hydrogel composition of claim 1,wherein the hemostatic hydrogel composition further comprises a ratio ofweight-average molecular weight (Mw) of the acrylated chitosan to theoxidized dextran from about 2 to about 10, a ratio of number-averagemolecular weight (Mn) of the acrylated chitosan to the oxidized dextranfrom about 4 to about 15, a ratio of z average molecular weight (Mz) ofthe acrylated chitosan to the oxidized dextran from about 2 to about 10,a ratio of polydispersity index (PDI) (Mw/Mn) of acrylated chitosan tooxidized dextran from about 0.5 to about 0.8, a ratio of PDI (Mz/Mw) ofacrylated chitosan to oxidized dextran from about 0.5 to about 1.0, or acombination thereof.
 5. The hemostatic hydrogel composition of claim 1,wherein the hemostatic hydrogel composition comprises a bound watercontent of from about 65% w/w to about 95% w/w.
 6. The hemostatichydrogel composition of claim 1, wherein the hemostatic hydrogelcomposition comprises a three-dimensional porous structure comprisinglayers of substantially non-interconnected pores having (a) a pore sizedistribution from about 10 μm to about 850 μm in diameter, (b) aplatelet adhesive surface, or a combination of (a) and (b).
 7. Thehemostatic hydrogel composition of claim 1, wherein the hemostatichydrogel composition comprises walls disposed between substantiallynon-interconnected pores, the walls having a wall thickness of from0.046 μm to 50 μm.
 8. The hemostatic hydrogel composition of claim 1,wherein the hemostatic hydrogel composition comprises a plateletadhesive surface so that, when in contact with blood, the hydrogelcomposition permits platelets, red blood cells, or a combination thereofwithin the blood to adhere to the platelet adhesive surface and promoteblood clot formation at or within the hydrogel composition.
 9. Thehemostatic hydrogel composition of claim 1, wherein the hemostatichydrogel composition comprises a platelet adhesive surface so that, whenin contact with blood, the hydrogel composition permits platelet, redblood cells, or a combination thereof within the blood to adhere to theplatelet adhesive surface and not permit platelets and/or red bloodcells from the blood to enter pores present in a first surface of thehydrogel composition, pass through the hydrogel composition, and thenexit the hydrogel composition via pores present in a second surface ofthe hydrogel composition that opposes the first surface.
 10. Thehemostatic hydrogel composition of claim 1 further comprising a burststrength of greater than 20 mmHg as determined using an ASTM F 2392-04protocol at about 10 seconds after formation.
 11. The hemostatichydrogel composition of claim 1 further comprising a burst strength ofgreater than about 35 mmHg as determined using an ASTM F 2392-04protocol at about two minutes after formation.
 12. The hemostatichydrogel composition of claim 1 further comprising an elastic modulus offrom about 500 Pa to about 5000 Pa at from about 10 seconds to about 80seconds after the formation of the hydrogel composition.
 13. Thehemostatic hydrogel composition of claim 1 further comprising acompression modulus of from about 3 kPa to about 250 kPa.
 14. Thehemostatic hydrogel composition of claim 1 further comprising an averageadhesion strength of from about 1.0 N to about 50.0 N, as determinedusing an ASTM F 2258-05 protocol.
 15. The hemostatic hydrogelcomposition of claim 1 further comprising a volume that does notincrease upon exposure to a physiological fluid or body fluid.
 16. Thehemostatic hydrogel composition of claim 1, wherein the hemostatichydrogel composition is substantially transparent when the hydrogelcomposition has a thickness of 2 mm to 10 mm.
 17. The hemostatichydrogel composition of claim 1 further comprising a therapeutic agent,a plurality of stem cells, or a combination thereof.
 18. The hemostatichydrogel composition of claim 1, wherein the hemostatic hydrogelcomposition has a gelation time from about 10 seconds to about 240seconds after contacting the acrylated chitosan composition with theoxidized dextran composition.
 19. The hemostatic hydrogel composition ofclaim 1 comprising: (a) from about 0.00 to about 0.3 mole fraction of afirst monomer of formula (I)

(b) from about 0.02 to about 0.7 mole fraction of a second monomer offormula (III),

and (c) from about 0.0 to about 0.8 mole fraction of a third monomer offormula (V),


20. The hemostatic hydrogel composition of claim 19, wherein thehemostatic hydrogel composition comprises from about 0.0 to about 0.26mole fraction of the first monomer of formula (I).
 21. The hemostatichydrogel composition of claim 19, wherein the hemostatic hydrogelcomposition comprises from about 0.03 to about 0.6 mole fraction of thesecond monomer of formula (III).
 22. The hemostatic hydrogel compositionof claim 19, wherein the hemostatic hydrogel composition comprises fromabout 0.04 to about 0.7 mole fraction of the third monomer of formula(V).
 23. The hemostatic hydrogel composition of claim 19, wherein thehemostatic hydrogel composition comprises no greater than about 0.2 molefraction of a fourth monomer of formula (IV)


24. The hemostatic hydrogel composition of claim 19, wherein thehemostatic hydrogel composition comprises no greater than about 0.65mole fraction of a fifth monomer of formula (VIII)


25. The hemostatic hydrogel composition of claim 19, wherein thehydrogel comprises a plurality of crosslinked moieties of formula (IX)


26. A method of preparing a hemostatic hydrogel composition of claim 1,the method comprising contacting an acrylated chitosan compositionproduced by dissolving chitosan in an acetic acid solution prior toacrylation and comprising: (i) from about 0.01 to about 0.3 molefraction of a first monomer of formula (I)

(ii) from about 0.3 to about 0.75 mole fraction of a second monomer offormula (II)

(iii) from about 0.2 to about 0.7 mole fraction of a third monomer offormula (III)

with an oxidized dextran composition comprising: (i) less than about 0.8mole fraction of a first monomer of formula (V)

and (ii) from about 0.1 to about 1.0 mole fraction of a second monomer,wherein the second monomer is selected from a monomer of formula (VI), amonomer of formula (VII) and a combination of formula (VI) and formula(VII)

thereby to produce the hemostatic hydrogel composition.
 27. The methodof claim 26, wherein the ratio of the viscosity of the acrylatedchitosan composition to the oxidized dextran composition ranges fromabout 100:1 to about 10,000:1.
 28. A method of reducing or stoppingblood loss at a location in a subject in need thereof, the methodcomprising forming at the location in the subject the hemostatichydrogel composition of claim 1 thereby to reduce or stop blood loss atthe location.
 29. The method of claim 28, wherein the hemostatichydrogel composition comprises one or more of the following features:(i) the hemostatic hydrogel composition adheres to a tissue surface atthe location, (ii) platelets, red blood cells, or a combination thereofpresent at the location adhere to surfaces of pores in the hemostatichydrogel, (iii) the hemostatic hydrogel composition promotes plateletadhesion and collection, platelet activation, and platelet aggregationand concentration at the location, (iv) the hemostatic hydrogelcomposition promotes a blood coagulation cascade to occur at thelocation, and (v) the hemostatic hydrogel promotes blood clot formationat the location.
 30. The method of claim 28, wherein the blood loss iscaused by trauma, abrasion, or surgical intervention at the location.31. The method of claim 30, wherein the surgical intervention is cardiacsurgery, cranial surgery, spinal surgery, OB-GYN surgery, organ surgery,plastic surgery, a sinus procedure, thoracic surgery, a vascularsurgery, a minimally invasive procedure, an arthroscopic procedure, anendoscopic procedure or a laparoscopic procedure.
 32. The method ofclaim 28, wherein the hemostatic hydrogel composition, when formed atthe location, is capable of filling a cavity at the location withoutinducing compression of tissue surrounding the cavity when thehemostatic hydrogel composition is exposed to physiological fluid or abody fluid.